The Body Condition index and Genetic connectivity among foraging Green Turtle Populations in Indonesia at different scales. A case study from Berau Green Turtle Rookery, East-Kalimantan.

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THE BODY CONDITION INDEX AND GENETIC CONNECTIVITY AMONG FORAGING GREEN TURTLE

POPULATIONS IN INDONESIA AT DIFFERENT SCALES :

A CASE STUDY FROM BERAU GREEN TURTLE ROOKERY, EAST-KALIMANTAN

Windia Adnyana

The Faculty of Veterinary Medicine, Udayana University, Bali - Indonesia

SUMMARY

The purpose of this study is to assess the body condition index of green turtles at three different seagrass

meadows of the Berau Isles, assess the genetic composition of green turtles, identify presence or absence of

genetic differentiation among the green turtles reside in the three foraging areas, and to identify links among

these foraging areas and various nesting sites in the Australasian region.

Sampling were carried out between 8 to 23 December 2009 from three main foraging locations within the Berau

Isles. The selected foraging habitats were the water in the northern side of Pulau Derawan, near Pulau Panjang,

and the water of Payung-Payung near Maratua Island. Turtles were captured either by net or rodeo technique,

taken onboard, their biometric values were measured and the skin samples were taken for genetic analysis.

A total of 310

Chelonia mydas

were captured and observed. Most of them (50.32%) are medium size turtles with

Curved Carapace Length between 60-80 cm. The proportions of turtle with CCL > 80 cm and with CCL < 60 cm

were 27.10% and 22.58%, respectively. Their bodyweight ranged from 7.70 kg to 158.90 kg. In view of their sex

status which was determined based on the Total Tail Length, 17.42% were defined to be males and 10.32% were

females. Most turtles (72.26%) were sexually undifferentiated by means of external characteristics. Thir Body

Condition Index (BCI) varied between 0.71

–

1.80. Most turtles were in very good (70.0%) and good (11.29%)

conditions, as compared to average (8.06%) and poor conditions (10.65%). Based on their capture locations, the

highest BCI was calculated for Payung-Payung population (1.30±0.21; range=0.80

–

1.66; n=102), followed by

Pulau Derawan (1.26±0.16; range=0.84

–

1.56; n=117) and Pulau Panjang population (1.24±0.20;

range=0.71-1.80; n=91). Statistical analysis showed that there was no significant difference between the population of Pulau

Panjang and Pulau Derawan (P=0.443). Significant difference (P=0.017) was found between the population of

Pulau Panjang and Payung-Payung., but not-significant difference was calculated between Payung-Payung and

Pulau Derawan (P=0.078).

A total of 213 mitochondrial (mt) DNA fragments, out of 309 collected samples were amplified by PCR technique.

Screening of polymorphism within 384 bp mtDNA control region fragments identified 34 polymorphic sites and 17

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2

(19.7%), A3 (12.2%), C5 (10.8%), and C14 (6.6%). These five variants accounted for 90.1% of the total resident

foraging population. The other haplotypes pesent in relatively small proportion, ranged from 0.5% to 1.9%.

Despite of minor variations, there was no significant difference (P>0.05) found on the genetic structure of resident

populations in the Pulau Panjang, Pulau Derawan, and Payung-Payung foraging habitats. Mixed stock analysis

(MSA) results revealed that the feeding populations of Berau water were mainly composed of green turtles from

the Turtle Islands Heritage Protected Area (TIHPA) (45.49%), the nesting populations of Berau rookery (26.82%),

Micronesia (9.3%) and Papua New Guinea (8.44%) nesting populations. Small proportion of representatives from

the nesting sites in the South China Sea regions and Aru were also found. The finding emphasizes the need to

build a network of turtle based-MPA across SSME - BSSE - and Micronesian regions.

The population structure of green turtles resided in these three foraging sites were similar, but individual

exchange among the foraging sites is unlikely, which implied that each feeding habitat should be managed

separately. The body condition index of green turtles from Pulau Panjang and Pulau Derawan were significantly

lower than their counterpart which were captured in Payung-Payung. This finding, perhaps, associated with the

relative distance of the foraging habitat to the river come from the mainland of Kalimantan. Payung-Payung is

relatively far away from the mainland as compared to the other two, and possibly the sedimentation substrate

which influences the fertility of the sea grass are present in lower concentration in this water. Managing the

cleanliness of the river will help in maintaining the fertility of the seagrass beds in Pulau Panjang and Pulau

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INTRODUCTION

The Berau Islands complex is an important nesting and foraging area for the green sea turtle (

Chelonia mydas

) in

Indonesia. This area is located within coordinates 02

0

49’42.6’’

- 01

0

2’0.06’’

N; 117

0

59’17.16’’

- 119

0

2’50.30’’

S and

belongs to the Berau district in East-Kalimantan (

Figure-1

). The area encompasses over 1.2 millions ha of

coastal area across 31 small islands, nine of which represent important nesting areas for green turtles. Annual

census surveys since 2002, suggest that the Berau Islands have one of the highest density of nesting green sea

turtles in the South-East Asia region (Adnyana

et al

, 2007). Additionally to nesting beaches, the Berau Isles also

contains several large seagrass meadows, which provide a critical foraging habitat for green turtles. Recent

surveys, conducted by researchers from the Radboud University in Nijmegen in collaboration with WWF

Indonesia and Udayana University, revealed that the density of green turtles on the seagrass meadows in this

area is among the highest in the world (Christianen in prep). The average green turtle density on the foraging

grounds is 17 ± 1.5 individuals per hectare.

The spatial extend of some of the seagrass meadows in Berau region is declining. The cause of this decline is

believed to be related to increased nutrient and sediment loads as a result of upstream erosion in the Berau river.

A reduced area of available nutrients could potentially lead to a reduced fitness of a green turtle population. The

resilience of a population under stress of e.g. seagrass loss or harvesting depends on the propensity of individual

turtles to switch foraging grounds and on the onset of the switching. Mark-recapture studies of foraging green

turtles from all size classes (Christianen in prep) suggest that subadult green turtles are not switching between

foraging grounds on a small spatial scale (i.e.within the Berau Isles) and thus, individuals associated with the

declining seagrass meadows are at risk of undernourishment. The body condition index (BCI) we be measured

to

infer food availability and quality at the different foraging areas within the Berau water.

Mark-recapture studies of adult green turtles have revealed links between the nesting and the foraging

populations within the Sulu-Sulawesi marine Ecoregion. For example, turtles that received a tag while nesting on

the beach of Palau, the Philippines, and Malaysia (Sarawak, and Sabah) were later recaptured while foraging at

Derawan, Panjang or Maratua waters (WWF pers. Comm.). In addition, some individuals were tracked using

satellite telemetry (for a maximum of 155 days) and found to migrate from the Derawan and Sangalaki Islands to

Sabah and the Philippines (Adnyana

et al

. 2007) after nesting. While these observations provide valuable insight

into individual movement patterns, the relatedness of the Derawan green turtle population to populations in other

parts of the Australasian region, as well as an understanding of genetic differentiation among feeding grounds

within the archipelago remains unresolved. An assessment of the genetic composition of the Berau foraging

population examines the relative contribution of green turtle breeding populations to this assemblage, thus

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This study is part of a PhD project on “

sea grasses

under green turtle grazing and nutrient loads”, conducted by

Marjolijn Christianen, Radboud University Nijmegen. This project primarily builds on previous genetic studies of

Australasian green turtle nesting populations, identifying 17 genetically distinct breeding stocks (Dethmers

et al

.

2006). This work is critical as the success of green turtle management strategies is contingent on understanding

of their population dynamics.

Objectives of this study

1.

Assess the body condition index of green turtles at three different seagrass meadows of the Berau Isles

2.

Assess the genetic composition of green turtles at the three main seagrass meadows of the Berau Isles

3.

Identify presence or absence of genetic differentiation among the three foraging areas.

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Figure-1:

A map showing the locations of three different foraging habitats within the Berau Archipelago. The

approximation of geographic location of this Isles is 02

0

49’42.6’’

- 01

0

2’0.06’’

N; 117

0

59’17.16’’

- 119

0

2’ 50.30’’

S.

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MATERIALS AND METHODS

Sample collection, measurements, maturity and sexual determination

Sampling were done during 15 working days, from 8 to 23 December 2009 by a team led by Marjolijn Christianen

,

a PhD candidate from Radboud University Nijmegen. All samples were taken from three main foraging locations

within the Berau Islands complex, i.e. the water in the northern side of Pulau Derawan, near Pulau Panjang, and

the water of Payung-Payung near Maratua Island (see

Figure-1

). Turtles were captured either by net or rodeo

technique (jumping straightly to the turtles), taken onboard, and mesured for their Curved Carapace Length

(CCL), Total Tail Length (TTL), and their body-weight (BW). All of these measurement were carried out following

procedures provided by Bolten (1999) and the protocol provided by Adnyana and Hitipeuw (2009).

In view of the maturity status, based on their CCL, turtles with a CCL <80 cm were considered as immature, while

those with a CCL >80 cm were defined as mature. This criteria is decided based on our field experiences while

observing nesting green turtles on Derawan and Sangalaki Islands in which their minimum CCL was 80 cm. The

sex was predicted by looking at the TTL. Turtles of all size classes with TTL >20 cm were defined as males, while

those with TTL up to 20 cm and the CCL measuring more than 80 cm were defined as females. Immature turtles

(CCL up to 80 cm) with TTL less than 20 cm were considered as unsexed or sexually undetermined.

The Body condition index (BCI), a variable to indicate a decline in individual and population health which needed

to inform inshore management strategies, calculated using Equation derived from Bjorndal

et al.

(2000). BCI

quantifies body condition based on a ratio of weight and SCL. The equation is BCI = ([Weight (kg) / SCL(cm)

3

] x

10000). The straight carapace length (SCL) did not measured during this study. It was estimated from the values

of the CCL by using an equation SCL = 15 + (0.76 X CCL). This equation was made based on field records

obtained from Derawan nesting island monitoring workers. Total data used for linear regression calculation was

285, and the coeficient correlation (R) and R

2

were 0.771 and 0.594, respectively.

Any observed external morphological lesions on the turtle skin, carapace and plastron such as presence of scars,

wound, notch on marginal scutes, algae, barnacles, and fibropapillomatosis were recorded. Presence of metal

tags in the pliffers were noted, and turtles without tag were given a new one following the protocol provided by

Balazs (1999). Prior to be released back to the water, from each captured turtles, skin samples were taken from

flippers or neck region by using a biopsy punch, and immediately stored in either alcohol 70% or a NaCl saturated

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Molecular Methods

Genomic DNA was isolated from 0.1 g of skin tissue using

Qiamp™ DNA Mini Kit from Qiagen

®, and stored at

-20

o

C for subsequent polymerase chain reaction (PCR).

Successful DNA isolation was confirmed by running 2 μL

of genomic DNA in an ethidium bromide added 1% Agarose gel. A 740 bp segments of the mtDNA control region

for all samples were amplified using

LTEi9 (5'-AGCGAATAATCAAAAGAGAAGG-3')

and

H950

(5'-GTCTCGGATTTAGGGGTTTG-3')

primers (Abreu-Grobois

et al

, 2006). The first primer

binds at the border

between the tRNA-Thr and tRNA-Pro loci, and the second at position 782 near the end of the d-loop, expected to

produce PCR products of about 860 base pairs (bp). The target mitochondrial sequences were amplified by PCR

using approximately 50 ng of template genomic DNA in 25

µl

reaction volume containing

14.3 µl H

2

O, 2.5 µl DNA

genome, 1.5 units ampli

Taq

gold polimerase (applied biosystem), 1.5 µl PCR buffer (applied biosystem), 2.5 µl

MgCl

2

25 Mm, 2 µl dNTP 1 Mm, 1 µl of each primer 10 Mm.

The PCR profile comprised an initial denaturation of

5 min at 94°C (to activate the Ampli

Taq

gold polymerase),

followed by 40 cycles of: 94

o

C for 45 sec

(denaturation), 55

o

C for 45 sec (annealing), 72

o

C for 45 sec (extension), ended in a final extension in 72

o

C for 4

min. Electrophoresis was performed to confirm the result of amplification and to determine the length of PCR

product. One µl loading dye (bromophenol-blue and cyline cyanol) was added in 2 µl PCR product.

Electrophoresis was run for negative and positive control (marker) for 30 minutes in 50 Volt of 1% agarose gel

media with etidum bromide dye. PCR product was sent to Macrogen Inc. (Korea) for forward and reverse

sequencing.

Statistical Analysis

Biometrics data were analysed using the Statistical Package for Social Sciences (SPSS) version 13.0. Graphical

presentations were completed either by Microsoft EXCEL version 2010 or SPSS version 13.0. Sequences were

aligned using Clustal X (Thompson

et al

1997) and the population parameters such as polimorphic sites, the

percentages of each haplotype, haplotype diversity, and nucleotide diversity were analyzed using DNAsp 4.10

(Rozas

et al

., 2003). Phylogenetic tree was constructed to visualize the relationship among the observed mt-DNA

variants. AMOVA (Excoffier

et al

1992), Exact tests of population differentiation (Raymond & Rousset 1995) and

pairwise Fst tests (Slatkin 1991) implemented in the population genetics package Arlequin version 3.01 (Excoffier

et al, 2006) were used to examined genetic structure among surveyed populations. Mixed stock analysis, to

predict the individual contribution from nesting colonies or management unit to the population of green turtles

reside in the foraging grounds of Pulau Panjang, Pulau Derawan, and Payung-Payung were calculated BAYES

program (Pella and Masuda, 2001).

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RESULTS

Biometrics, maturity and sex status of Green Turtles in Berau Foraging Grounds

There was a total of 310 individual

Chelonia mydas

captured during 15 days sampling period during 8 December

–

23 December 2009. Their Curved Carapaca Length (CCL) encompassed all size classes (

Figure-2

) from small

immatures (CCL=40.80 cm) to large adults (CCL=111.30 cm). The mean ± standard deviation of the CCL was

71.69 ± 15.30 cm. When the CCL values were pooled into three categories (

Figure-3

), the majority of samples

were found to be medium size with CCL between 60-80 cm (50.32%). The proportions of large-adult (CCL > 80

cm) and small immature turtles with CCL < 60 cm were 27.10% and 22.58%, respectively.

Based on their actual capture sites, the percentage of mature-adult turtles (CCL>80 cm) was proportionally

highest in the Payung-Payung foraging ground as compared to the population of Pulau Panjang and Pulau

Derawan (

Table-1

). Consequently, as shown in

Figure-4

, the highest mean value for CCL was also found in the

foraging population of Payung-Payung (75.35±16.90 cm; range = 43.50

–

111.30 cm; n = 102), followed by Pulau

Panjang (70.63±15.02; range = 40.80

–

102.40 cm; n = 91) and the foraging population of Pulau Derawan

(69.32±13.46; range = 46.80

–

104.50 cm; n = 91). Statistically, a one way anova test suggested that significant

differences were calculated between the mean value of CCL of Payung-Payung population and the other two

populations, i.e. Pulau Panjang (P=0.031) and Pulau Derawan (P=0.003). However, no significant difference was

calculated between Pulau Panjang and Pulau Derawan (P=0.535).

Table-1

: Size classes indicated by the Curved Carapace Length (CCL) of the

Chelonia mydas

captured in three

foraging grounds of the Berau Isles.

Capture Locations Size Class Total

>80 cm 60-80 cm <60 cm

Pulau Panjang 21 (23.08%) 51 (56.04%) 19 (20.88%) 91 (100%)

Pulau Derawan 24 (20.51%) 64 (54.70%) 29 (24.79%) 117 (100%)

Payung-Payung 39 (38.24%) 41 (40.20%) 22 (21.57%) 102 (100%)

Combined 84 (27.10%) 156 (50.32%) 70 (22.58%) 310 (100%)

A similar trend was observed for the turtle bodyweight which varied between 7.70 kg to 158.90 kg (

Figure-5

).

Overral, the mean ± standard deviation was 48.75±29.65 kg. In view of the actual capture sites, due to its class

size distribution as shown in

Table-1

, the highest mean value for the body weight was observed in

Payung-Payung turtle foraging population (57.36±34.73 kg; range = 8.90-158.90 kg; n = 102), much higher as compared

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Payung-9

Payung population and the other two populations, i.e. Pulau Panjang (P=0.007) and Pulau Derawan (P=0.000).

No significant difference was calculated between Pulau Panjang and Pulau Derawan (P=0.556).

In view of their sex status which was determined based on the Total Tail Length, 17.42% were defined to be

males and 10.32% were females. Most turtles (72.26%) were sexually undifferentiated. Two turtles with CCL

between 74 and 75 cm were classified as males as their TTL > 20 cm. The summary values for maturity and sex

status per capture locations were presented in

Table-2

.

Figure-2:

Histogram showing frequency distribution of the curved carapace length (CCL) of 310 green sea turtles

captured, tagged and measured in three foraging grounds of Berau water. Normal curve is displayed.

120.00 100.00

80.00 60.00

40.00

CCL

40

30

20

10

0

Fre

qu

en

cy

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Figure-3

: Size class distribution of green sea turtles captured in three different locations in Berau water.

Figure-4:

The 95% confidential interval of the CCL of green turtles captured in three different foraging grounds in

the Berau water. Noted that, the highest mean value for CCL was in the foraging population of Payung-Payung,

followed by Pulau Panjang and the foraging population of Pulau Derawan. See the associated text for further

description.

<60 cm 60-80 cm

>80 cm

Maturity

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0%

P

erc

en

t

22.58% 50.32%

27.1%

Payung-Payung Pulau Derawan

Pulau Panjang

Locations

80

78

76

74

72

70

68

66

95

%

C

I C

C

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Figure-5:

Histogram showing frequency distribution of the body weight of 310 green sea turtles captured in three

foraging grounds of Berau water.

Figure-6:

The 95% confidential interval of the bodyweight of green turtles captured in three different foraging

grounds in the Berau water. Note that, the highest mean value was in the foraging population of Payung-Payung,

followed by Pulau Panjang and the foraging population of Pulau Derawan. See text for further description.

200.00 150.00

100.00 50.00

0.00

Bodyweight

70

60

50

40

30

20

10

0

Fre

qu

en

cy

Mean = 48.7465 Std. Dev. = 29.65113 N = 310

Pulau Panjang

Locations

65

60

55

50

45

40

35

95

%

C

I B

od

ywe

igh

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12

Table-2

: Maturity and sex status green turtles sampled in Berau waters (N=310). Noted that males were defined

when the Total Tail Length (TTL) of any size class of turtles was more than 20 cm. Mature turtles were those who

have the Curved Carapace Length (CCL) of more than 80 cm.

The Body Condition Index (BCI) of all samples varied between 0.71

–

1.80, normally distributed (

Figure-7

) with

the smallest and the highest values were both found in turtles captured from Pulau Panjang. Based on the criteria

defined by Bjorndal et al (2000) (

Table-3

), it was found that (

Figure-8)

most turtles were in very good (70.0%)

and good (11.29%) conditions, as compared to average (8.06%) and poor conditions (10.65%).

Regarding their size classes, as shown in

Figure-9

, the highest mean value for BCI (1.44±0.12; range =

1.14-1.66; n = 84) was noted in mature-adult turtles measuring > 80 cm. The mean values for size classes between

60-80 cm and <60 cm were 1.28±0.11 (range=0.88-1.60-80; n=156) and 1.02±0.13 (range=0.71-1.44; n=70),

respectively. The BCI mean values were calculated significant across the size classes (P=0.000).

Figure-7

: Histogram showing frequency distribution of the body weight of 310 green sea turtles captured in three

foraging grounds of Berau water. Normal curve is added.

Male

Female

Male

Female

Undetermined

Pulau Panjang

12 (13.19%) 9 (9.89%)

- 70 (76.92%)

-

91 (100%)

Pulau Derawan

16 (13.68%) 8 (6.84%)

- 93 (79.49%) 117 (100%)

-Payung-Payung

24 (23.53%) 15 (14.71%) 2 (1.96%) - 61 (59.80%) 102 (100%)

Combined

52 (16.77%) 32 (10.32%) 2 (0.65%)

224 (72.26%) 310 (100%)

-Capture Locations

Mature (CCL >80 cm)

Immature (CCL < 80 cm)

Total

2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60

Body Condition Index

50

40

30

20

10

0

Fre

qu

en

cy

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Table-3:

Body condition index corresponding to subjective visual observations in Green turtles (

Chelonia mydas

).

Calculation was made using Equation derived by Bjorndal

et al.

(2000). BCI quantifies body condition based on a

ratio of weight and SCL. The equation is BCI = ([Weight (kg) / SCL(cm)

3

] x 10000).

Condition Index

Code

Body Condition

Index

Subjective Visual Interpretation

0

> 1.20

Very Good

1

1.11

–

1.20

Good

2

1.00

–

1.10

Average

3

< 1.00

Poor

Determined from data presented by Bjorndal, K. A., Bolten, A. B. and Chaloupka, M. Y. (2000). Green turtle

somatic growth model: evidence for density dependence’.

Ecological Applications

10, 269-282.

Figure-8

: The Body Condition Index (BCI) of all samples captured from three foraging sites in the Berau water.

Note that most turtles were in very good and good conditions, as compared to average and poor conditions.

Poor Average

Good Very good

Body Condition Index

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0%

Perc

en

t

10.65% 8.06%

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Figure-9:

The 95% confidential interval of the body condition index for various size classes of green turtles

captured in Berau water. See text for further description.

Based on their capture locations, the summary of the body condition of the samples is shown in

Table-4

. Overall,

the mean value (±standard deviation) for the BCI in Payung-Payung population (

Figure-10

) was the highest

(1.30±0.21; range=0.80

–

1.66; n=102), followed by Pulau Derawan (1.26±0.16; range=0.84

–

1.56; n=117) and

Pulau Panjang population (1.24±0.20; range=0.71-1.80; n=91). Statistical analysis showed that there was no

significant difference between the population of Pulau Panjang and Pulau Derawan (P=0.443) but significant

(P=0.017) with the population of Payung. Non-significant difference was also calculated between

Payung-Payung and Pulau Derawan (P=0.078).

Table-4:

The summary of the body condition index (BCI) of green sea turtles captured in three foraging locations

in Berau water.

<60 cm 60-80 cm

>80 cm

Maturity

1.5

1.4

1.3

1.2

1.1

1.0

0.9

95

%

C

I B

od

y

Con

di

tion

Ind

ex

Very Good

Good

Average

Poor

Pulau Panjang

65 (71.43%)

6 (6.59%)

6 (6.59%) 14 (15.38%)

91 (100%)

Pulau Derawan

75 (64.10%) 23 (19.66%) 12 (10.26%) 7 (5.98%)

117 (100%)

Payung-Payung

77 (75.49%)

6 (5.88%)

7 (6.86%) 12 (11.76%) 102 (100%)

Combined

217 (70%)

35 (11.29%) 25 (8.06%) 33 (10.65%) 310 (100%)

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Figure-10:

The 95% confidential interval of the body condition index (BCI) of green turtles captured in three

different foraging grounds in the Berau water. Note that, the highest mean value was in the foraging population of

Payung-Payung, followed by Pulau Derawan and the foraging population of Pulau Panjang. See text for further

description.

The genetic composition of green turtles at the three main seagrass meadows of the Berau Isles

A total of 213 mitocondrial (mt) DNA fragments, out of 309 collected samples of green sea turtles captured from

Berau water were amplified by PCR technique. Our screening of polymorphism within 384 bp mtDNA control

region fragments identified 34 polymorphic sites (

Table-5

) and 17 distinct haplotypes, the frequencies of which

are shown in

Table-6

. Regardless the actual locations of the foraging habitats, the most frequent haplotype

identified in Berau waters was D2 (40.8%), followed by C3 (19.7%), A3 (12.2%), C5 (10.8%), and C14 (6.6%).

These five variants accounted for 90.1% of the total resident foraging population. The other haplotypes pesent in

relatively small proportion, ranged from 0.5% to 1.9%.

Note: siryngodium vs enhalus sp

Pulau Panjang

Locations

1.35

1.30

1.25

1.20

95

%

C

I B

od

y C

on

di

tion

Ind

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16

Table-5

: Number of polymorphic sites, total haplotypes, haplotypes diversity (Hd) and nucleotide diversity (π) of

mt-DNA sequences found in three foraging locations of Berau waters. Screening of polymorphism was done

within 384 bp mtDNA control region fragments.

Table-6:

Frequency of mt-DNA haplotypes found in three foraging locations of Berau waters. Screening of

polymorphism was done within 384 bp mtDNA control region fragments.

The haplotypes identified in this study can be broadly group into three categories (

Figure-11

and

Table-6

). The

first is those that are widespread occurring in all three surveyed foraging habitats in high (D2, C3, A3, C5, and

C14) and low frequencies (C4). The second is those which are found in two foraging grounds in relatively low

frequencies (A1, E2, and A4), and the third is those that are identified only in one foraging ground (A6, B4, C7,

New1, New2, New3/P1, New4, and New5).

Figure-11

: Frequencies of green sea turtle haplotypes identified in three foraging grounds in Berau, East

Kalimantan of Indonesia. Data refered to

Table-6

.

Hd Sd π Sampling Sd

Pulau Panjang 91 60 25 11 0.780 0.039 0.02048 0.00344

Derawan North 116 91 33 12 0.753 0.034 0.01430 0.00286

Payung-Payung 102 62 27 9 0.767 0.037 0.02419 0.00304

Overall Berau Waters 309 213 34 17 0.762 0.021 0.01918 0.00188

Haplotype diversity Nucleotide diversity

Locations of the foraging grounds

total mtDNA

fragments Total haplotypes

Number of polymorphic (segregating) sites Total colected

samples

C3 C4 C5 C7 C14 D2 B4 A1 A3 A4 A6 E2 New1 New2 New3 New4 New5

Pulau Panjang 12 2 6 1 3 24 8 1 1 1 1 60

Pulau Derawan 20 1 12 7 38 1 2 5 2 1 1 1 91

Payung-Payung 10 1 5 4 25 2 13 1 1 62

42 4 23 1 14 87 1 4 26 2 1 3 1 1 1 1 1 213

19,7 1,9 10,8 0,5 6,6 40,8 0,5 1,9 12,2 0,9 0,5 1,4 0,5 0,5 0,5 0,5 0,5 100,0 %

Total

Feeding Habitats

Total

(17)

17

Five variants, i.e. three were identified in Pulau Derawan foraging ground and one each for Pulau Panjang and

Payung-Payung are assigned as new variants as they have not been described previously in the Australasian

region by Moritz

et al

(2002). The haplotype of new3 identified in the water near Pulau Panjang has been

confirmed to be similar to a haplotype that occur in low frequency in Paka nesting population (which is assigned

as P1) of Peninsular Malaysia (Wahidah, pers comm).The sequences for all of these variants are shown in

Annex-1.

Incorporation of the haplotypes of green turtles identified in this work into the Neighbour-Joining tree of genetic

structure of Australasian green turtle (

Figure-12

) constructed by Dethmers

et al

(2006) showed that two clades

(clade I and V) were strongly represented in the population of green turtles resided in Berau waters. Total green

sea turtles having alleles that belongs to clade I were 174 (81.69%) and clade V were 38 (17.84%). Single

(0.47%) green turtle was identified carrying haplotype B4 which belongs to clade II.

(18)

18

identified by Dethmers

et al

(2006). Haplotype nomenclature follows Norman

et al

(1994). Suk (Sukamade of East

Java), PJ (Pulau Panjang), PY (Payung-Payung), and D (Pulau Derawan). The variant of New3 has been

confirmed identical with the haplotype P1 which has been identified to occur in low frequency in Paka nesting

population of Peninsular Malaysia (Wahidah, pers comm).

Genetic differentiation among the three resident populations in the Berau foraging areas

The genetic structures of the feeding populations of the Pulau Panjang, Pulau Derawan, and Payung-Payung are

presented in

Figures 13-15

. Despite of minor variations, Exact Test of sample differentiation based on haplotype

frequencies among the three foraging habitats in Berau water revealed that all of the pairwise comparisons were

non-significant (P>0.05). Similar result was also obtained from pairwise Fst tests which showed no significant

different (P>0.05) on the genetic structure among the surveyed resident populations of the Pulau Panjang, Pulau

Derawan, and Payung-Payung foraging habitats.

(19)

19

Figure-14

: Proportion (%) of haplotypes identified in the foraging habitat of Pulau Derawan, Berau of East

Kalimantan

–

Indonesia (N=91).

Figure-5

: Proportion (%) of haplotypes identified in the foraging habitat of Payung-Payung, Berau of East

Kalimantan

–

Indonesia (N=62).

Possible Contributing Stocks for the foraging areas of Berau waters

In the foraging ground of Pulau Panjang, apart from the presence of C3 which is not particularly diagnostic given

its wide distribution in the Australasian green turtle rookeries, the other haplotypes are indicative. The

combination of haplotypes D2, C5, and C14 in high frequencies suggested the importance of rookeries in the

(20)

20

Ampat

(Papua),

Micronesia

, and perhaps

Ashmore Reef

. C4 is a dominant haplotype for

Sarawak

breeding

population and found in low frequency in the nesing colonies of

Redang Island

(Peninsular Malaysia) and the

Northern Great Barrier Reef (Australia). Nevertheless, the absence of haplotype B1, excluded the possibility of

Northern Great Barrier Reef MU as a source of nester. The low frequencies of the haplotypes of C7, A6, and

New3/P1 were only identified in this foraging ground but absent from the feeding habitats of Pulau Derawan and

Payung-Payung. While C7 confirmed the representation of Long Island rookery, A6 and New3/P1 are diagnostic

for the nesting colony of Sangalaki Island (East Kalimantan, Indonesia) and

Paka

(Peninsular malaysia),

respectively.

Similarly with the population of Pulau Panjang foraging ground, the most frequent variant in Pulau Derawan

feeding population was D2 (41.8%), folowed by C3 (22.0%), C5 (13.2%), and C14 (7.7%), implying that the

Sulu-Sulawesi Seas rookeries

and

Aru

nesting colony are a major and strongly represented in this population.

Presence of A3 (5.5%), A1 (2.2%) and E2 (2.2%) indicated contribution from

Micronesian

,

PNG

,

Raja Ampat

,

and

Ashmore Reef

breeding aggregates. Similarly to Pulau Panjang assemblage, presence of C4 indicated the

contribution from

Sarawak

and

Redang

nesting populations. Finding of B4 in low frequency in this feeding site is

intriguing, and possibly represented the nesters from

Khram Island

(Gulf of Thailand) and/or

Lacepede Island

of

North West Shelf (Australia). A single haplotype of New1 is found to be similar to Pi41 which is diagnostic for Raja

Ampat nesting population (Velez-Zuazo et al, 2008). The other two new variants, New2 and New, were orphan

haplotypes in which the contributors could not be identified at the present study.

Like the other foraging habitats (Pulau Panjang and Pulau Derawan), the resident population in this water was

dominated by the haplotype of D2 (40.3%), A3 (21.0%), C3 (16.1%), C5 (8.1%), C14 (6.5%), and A1 (3.2%). The

variants, i.e. A4, C4, and New4 were found in very low frequencies (1.6% each). Apart from the orphan haplotype

of New4, the others represented green turtle nesting assemblages from the Sulu-Sulawesi Seas Rookeries as the

primary contributors, as well as, breeding colonies from Micronesia, PNG, Raja Ampat

–

Papua, Ashmore Reef

(Australia), Aru, Sarawak and/or Redang Island of Peninsular Mlaysia.

Overall, the mixed stock analysis (MSA) results confirmed that the feeding populations of Berau water were

mainly composed of the Turtle Islands Heritage Protected Area (TIHPA) turtles that belongs to Malaysia and

Philippines (45.49%) and the nesting populations of Berau rookery (26.82%). The Micronesia and Papua New

Guinea nesting populations were represented at 9.3% and 8.44%. Other rookeries, such as Sarawak, Aru, and

Pangumbahan of West Java were represented at 1.45%, 2.12%, and 5.41%, respectively (

Table-7)

. The high

proportions of turtles from TIHPA and Berau MUs among the foraging turtles is expected given the proximity of

(21)

21

Table-7:

Proportions of green turtle nesting populations from various reookeries within the Australasian region

which identified in the feeding grounds of Berau waters are presented in

Table-7

.

Contributing Stocks

(Management Units)

Pulau Derawan

Payung-Payung

Pulau Panjang

Overall (Berau)

1

Northern Great Barrier Reefs

0

2

Coral Sea

0

3

Southern Great Barrier Reefs

0

4

New Caledonia

0

5

Micronesia

9,88

11,52

6,57

9,3

6

Papua New Guinea

0

15,67

12,93

8,44

7

Gulf of Carpentaria

0

8

Aru

3,09

3,34

0,12

2,12

9

Berau Islands

28,5

18,6

29,6

26,82

10

South-East Sabah

0,94

2,84

3,88

0,6

11

Sulu Sea

47,15

44,93

41,62

45,49

12

Sarawak

0

1,65

3,65

1,45

13

Peninsular Malaysia

0

14

Ashmore Reef

0

0,01

0

15

Scott Reef

0

16

West Java

9,33

1,44

1,63

5,41

17

North West Shelf

1,11

0

0,37

(22)

22

CONCLUSION AND MANAGEMENT IMPLICATION

This study confirmed that the foraging grounds of Pulau Panjang, Pulau Derawan, and Payung-Payung are

genetically the most diverse sample analysed so far within the Australasian region, indicating the presence of

several green turtle stocks at high and low frequencies. Most turtles represented rookeries from the Malaysian

and Philippines Turtle Islands (45.49%), Berau nesting islands (26.82%), Micronesia (9.3%), and The birdhead of

Papua/Papua New Guinea (8.44%). Small proportion of green turtles were also representing nesting sites in the

South China Sea regions, and Aru. This finding emphasizes the need to build a network of sea turtle management

across SSME - BSSE - and Micronesian regions.

The population structure of green turtles resided in these three foraging sites were similar. Nevertheless,

tag-recaptured study indicated that there was no individual exchange among the foraging sites (Christianen in prep).

This implied that each feeding habitat should be managed separately.

The body condition index (BCI) of green turtles from all three feeding sites were relatively 'very good'. however,

when comparation among the three sites was made, the BCI value of green turtles captured in the water of Pulau

Panjang and Pulau Derawan were significantly lower than their counterpart which were captured in

Payung-Payung. This finding, perhaps, associated with the relative distance of the foraging habitat to the river come from

the mainland of Kalimantan. Payung-Payung is far away from the mainland as compared to the other two, and

possibly the sedimentation substrate which influences the fertility of the sea grass are present in lower

concentration in this water. Managing the cleanliness of the river will help in maintaining the fertility of the

seagrass beds in Pulau Panjang and Pulau Derawan.

ACKNOWLEDGEMENT

This project was strongly supported by many people. We would like to express our sincere thanks to Rusli Andar

and his team, Hidayatun Nisa Purwanasari, and Made Jayaratha for their invaluable help during the field and lab

(23)

23

REFERENCES

Abreu-Grobois FA, Horrocks JA, Formia A et al (2006) New mtDNA dloop primers which work for a variety of

marine turtle species may increase the resolution capacity of mixed stock analyses. Poster presented at the 26th

Annual Symposium on Sea Turtle Biology and Conservation, Crete, Greece, 2-8 April 2006. Available from:

http://www.iucnmtsg

.org/genetics/meth/primers/abreu_grobois_etal_new_dloop_primers.pdf

Adnyana W, LP Soede, G Gearheart and M Halim (2007). Status of green turtle (

Chelonia mydas

) nesting and

foraging populations of Berau, East Kalimantan, Indonesia, including results from tagging and telemetry.

IOTN

(7), 1-11.

Adnyana W and C Hitipeuw (2009). Panduan Melakukan Pemantauan Populasi Penyu di Pantai Peneluran di

Indonesia. Edisi Mei, WWF Indonesia Marine Program, 31 pp.

Bolten AB (1999). Techniques for Measuring Sea Turtles. In Research and Management Techniques for the

Conservation of Sea Turtles, Eds. KL Eckert, KA Bjorndal, FA Abreu-Grobois, M Donnelly. IUCN/SSC Marine

Turtle Specialists Group Publication No. 4, p 110-114.

Bjorndal, K. A., Bolten, A. B. and Chaloupka, M. Y. (2000). Green turtle somatic growth model: evidence for

density dependence’. Ecological Applications

10, 269-282.

Dethmers K, D Broderick, C Moritz, NN FitzSimmons, CJ Limpus, S Lavery, S Whiting, M Guinea, RIT Prince, R

Kennett (2006). The genetic structure of Australasian green turtles (

Chelonia mydas

): exploring the geographical

scale of genetic exchange.

Molecular Ecology

, 15:3931-3946.Excoffier

et al

1992

Excoffier, L., P.E.Smouse and J.M.Quattro 1992. Analysis of molecular variance inferred from metric distances

among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479-491.

Excoffier L, G Laval, and S Schneider (2006). Arlequin ver 3.01 User Manual: An Integrated Software Package for

Population Genetics Data Analysis. Computational and Molecular Population Genetics Lab (CMPG). Institute of

Zoology, University of Berne, Baltzerstrasse 6, 3012 Bern, Switzerland. 143 pp.

Pella, J.J. and M. Masuda. 2001. Bayesian Methods for Analysis of Stock Mixtures from Genetic Characters. Fish

Bull 99:151-67

Raymond, M. and F.Rousset 1995 An exact test for population differentiation. Evolution, 49, 1280-1283.

Rozas, J., JC Sánchez-DelBarrio, X Messeguer, and R Rozas (2003). DnaSP, DNA polymorphism analyses by

the coalescent and other methods.

Bioinformatics

19: 2496-2497Slatkin 1991

Slatkin, M. 1991. Inbreeding coefficients and coalescent times. Gent. Res. Camb., 58, 167-175.

Thompson, J.D., T.J.Gibson, F.Plewniak, F.Jeanmougin and D.G.Higgins 1997. The Clustal X windows interface:

flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research,

24:4876-4882.

(24)

24

ANNEX-1:

Sequences from identified from green turtles captured in three different foraging grounds in

Berau waters during December 2009.

>W10198

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

* >W10200

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

* >W10201

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATTCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

* >W10202

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTGCATAAACATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTCACAGTAATTGGTTATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTCAATTTGTGGCGTACATAA TTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAGATTTATAACCT

* >W10203

* >W10204

* >W10205

*

>W10206

(25)

25

CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10207

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10208

*

>W10209

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTAAATTTGCATAAATGTTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTTACGGTAATTGGTTATTTCT TAAATAACTATTCACGAGAAATAAGCAACCCTTGTTGGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA TTTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTTCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10211

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTACATAAA-CATTTTAATAACATGAATATTAAGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTG TCACAGTAATTGGTTATTTCTTAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCA TTCAATTTGTGACGTACATAATTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCT TTAAAAGGCCTTTGGTTGAATGAGTTCTATACATTAGATTTATAACCT

*

>W10212

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10213

*

>W10214

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

(26)

26

*

>W10216

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTCATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10217

* >W10218

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAA-TATTTTAATAACATGAATATTAAGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCG TCACAGTAATTGGTTATTTCTTAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCA TTTAGTTTATAGCGTACATAACCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCT TTAAAAGGCCTTTGGTTGAATGAGTTCTATACATTAAATTTATAACCT

* >W10219

* >W10220

* >W10221

* >W10222

*

>W10223

(27)

27

TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10224

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10225

*

>W10226

*

>W10227

*

>W10228

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTACATAAACATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTCACAGTAATTGGTTATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTCAATTTGTGACGTACATAA TTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAGATTTATAACCT

*

>W10229

*

>W10230

*

(28)

28

*

>W10232

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

>W10233

*

>W10234

*

>W10236

*

>W10235

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAAATTTATAACC

* >W10238

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAAATTTATAACC

* >W10240

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATTCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAAATTTATAACC

(29)

29

* >W10246

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAAATTTATAACC

* >W10247

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTGCATAAACATTTTAACAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTCACAGTAATTGGTTATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTCAATTTGTGGCGTACATAA

TTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAGATTTATAACC *

>W10248

* >W10253

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATTCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA

CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGC---CTTTGGTTGAATGAGTTCTATACATTAAATTTATAACC *

>W10254

* >W10256

* >W10260

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTGCATAAACATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTCACAGTAATTGGTTATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTCAATTTGTGGCGTACATAA

TTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAGATTTATAACC *

>W10261

(30)

30

>W10263

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATTCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA

>W10265

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA

>W10266

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATTCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA

>W10270

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAAATTTATAACC

* >W10272

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATTCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAAATTTATAACC

* >W10277

* >W10279

* >W10282

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCC---TTTGGTTGAATGAGTTCTATACATTAAATTTATAACC

* >W10283

(31)

31

* >W10286

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTACATAAACATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTCACAGTAATTGGTTATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTCAATTTGTGGCGTACATAA TTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGCCT TTGGTTGAATGAGTTCTATACATTAGATTTATAACC

* >W10287

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTACATAAACATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTCACAGTAATTGGTTATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTCAATTTGTGGCGTACATAA TTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGCCT TTGGTTGAATGAGTTCTATACATTAGATTTATAACC

* >W10289

* >W10292

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACC

* >W10239

*

>W10245

*

>W10249

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAGTTTGCATAAACATTTTAATAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATTGTCACAGTAATTGGTTATTTCT TAAATAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAAGCCCATTCAATTTGTGGCGTACATAA TTTGATCTATTCTGGCCTCTGGTTGTTCTTTCAGGCACATATAAATAACGACGTTCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAGATTTATAACCT

*

>W10251

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATTTAGTTTATAGCGTACATAA CCTGATCTATTCTGGCCTCTGGTTGTCTTTTCAGGCACATACAAATAGTAACGTCCATTCGTTCCTCTTTAAAAGGCCTTTGGTTGAAT GAGTTCTATACATTAAATTTATAACCT

*

(32)

32

TAGCATATGACCAGTAATGTTAACAGTTGATTTGGCCCTAAACATGAAAATTATTGAATCCACATAAATATTTTAGTAACATGAATATTA AGCAGAGAATTAAAAGTGAAATGATATAGGACATAAAATTAAACCATTATACTCAACCATGAATATCGTCACAGTAATTGGTTATTTCT TAAGTAGCTATTCACGAGAAATAAGCAACCCTTGTTAGTAAGATACAACATTACCAGTTTCAGGCCCATT