DAmorocho_ PhD thesis

Foraging Ecology and Population Structure of the Green Sea Turtle

(Chelonia mydas) in the Eastern Pacific Coast of Colombia.

A thesis submitted for the degree of Doctor of Philosophy

Diego Fernando Amorocho Llanos

M.Sc.

School of Biological Sciences

Monash University

Melbourne, Australia

February 2009

Green sea turtle (Chelonia mydas) in the eastern Pacific island of Gorgona National Park,

Colombia

TABLE OF CONTENTS

LIST OF FIGURES i

LIST OF TABLES ii

ABSTRACT iii

GENERAL DECLARATION vi

ACKNOWLEDGEMENTS vii

CHAPTER 1. GENERAL INTRODUCTION

1.1 Green sea turtles Chelonia mydas agassizii in the eastern Pacific 1

1.2 Feeding and developmental grounds for green turtles 2

1.3 Nutritional ecology and importance of transitional habitats for green

turtles

3

1.4 Genetic composition of feeding grounds 4

1.5 Aims of the study 5

1.6 Thesis structure 6

CHAPTER 2. FEEDING ECOLOGY OF THE EASTERN PACIFIC

GREEN SEA TURTLE CHELONIA MYDAS AGASSIZII AT GORGONA

NATIONAL PARK, COLOMBIA.

Declaration for thesis chapter 2 7

Abstract 9

2.1 Introduction 9

2.2 Materials and methods 10

2.2.1 Study area 10

2.2.2 Sampling and content analysis 11

2.2.3 Laboratory analysis 11

2.2.4 Statistical analysis 12

2.3 Results 12

2.3.1 Diet composition 12

2.3.2 Dietary components and type of habitat 13

2.4 Discussion and conclusions 14

CHAPTER 3. DIGESTA COMPOSITION, INTAKE PASSAGE TIME

AND DIGESTIBILITY IN CAPTIVE EAST PACIFIC GREEN

TURTLES (CHELONIA MYDAS AGASSIZII) AT GORGONA

NATIONAL PARK, COLOMBIAN PACIFIC.


Abstract 17

3.1 Introduction 17


3.2.1 Study site 18

3.2.2 Turtles sampled, confinement and faecal collection 19

3.2.3 IPT and dietary treatments 19

3.2.4 Digesta composition and digestibility analyses 19

3.3 Results 20

3.3.1 Animal size and gut length 20


3.3.3 Digesta composition and digestibility analyses 20

3.4 Discussion 21

3.4.1 Turtle size class 21


3.4.3 Digesta composition 22

3.4.4 Digestibility 22

3.4.5 Comparisons with ruminants 22

3.4.6 Temperature implications 22

3.4.7 Conservation and management issues 23

3.4.8 Conclusions 23

CHAPTER 4. STOCK COMPOSITION OF THE GREEN SEA TURTLE

(CHELONIA MYDAS) IN EASTERN PACIFIC FORAGING GROUNDS

OF GORGONA NATIONAL PARK IN COLOMBIA.


Abstract 26

4.1 Introduction 27


4.2.1 Study site and sample collection 32

4.2.2 DNA extraction, PCR amplification and sequencing 32

4.2.3 Mitochondrial DNA haplotype characterization and data analysis 33

4.3 Results 34

4.3.1 Haplotypes identification 34

4.3.2 mtDNA genetic diversity and phylogenetic stock composition 35

4.4 Discussion 37

4.4.1 Genetic composition and diversity of Gorgona’s stock 37

4.4.2 Dispersal, recruitment and migratory behaviour of Gorgona stock 38

4.4.3 Conservation implications 40

CHAPTER 5. GENERAL DISCUSSION AND CONCLUSIONS 59

5.1 Shifting feeding diets 59

5.2 Nutritional benefits of an omnivorous diet 60

5.3 Gorgona: a stopover and marine protected area 61

5.4 Recommendations for further research 63

REFERENCES 64

i

LIST OF FIGURES

Fig. 2.1 Map of the study area in the Colombian Pacific 11

Fig. 2.2 Straight carapace length (SCL) distribution of captured turtles 12

Fig 2.3 Multiple means comparison of dietary components 14

Fig. 3.1 Map of Gorgona National Park in the Colombian Pacific 18

Fig. 3.2 In-water enclosure to keep turtles near shore El Poblado beach 19

Fig. 3.3 Percentage of markers recovered and time elapsed 20

Fig. 3.4 Percentage of different categories of ingested items collected from

faecal samples

20

Fig. 3.5a Pieces of mangrove fruit collected in faeces 21

Fig. 3.5b Leaves of Ficus spp collected in faeces 21

Fig. 4.1 Differences in colour and carapace shape of green sea turtle

juveniles corresponding to western CMP21 (GPC6) and eastern Pacific

CMP4 (GPC1) haplotypes recruited at Gorgona National Park in

Colombia.

51

Fig. 4.2 Gorgona National Park in the Colombian Pacific. 52

Fig 4.3 Maximum Parsimony phylogenetic tree of Gorgona’s green turtle

stock haplotypes.

53

Fig 4.4 Most parsimonious tree for sequences of haplotypes found in

Gorgona GPC (underlined) with their GeneBank assignation (CMP, E).

54

ii

LIST OF TABLES

Table. 2.1 Frequency and % dry mass of dietary components 12

Table 2.2 Nutritional contribution of dietary components 13

Table 2.3 ANOVA of seasonal grouped components 13

Table 3.1 Total mass of protein and plant feeding rations supplied 19

Table 3.2 Recovery of administered markers 20

Table 3.3 Percentages of organic matter, neutral detergent fibre, acid detergent

fibre, lignin and protein

20

Table 3.4 Number of days that turtles were confined, total food intake, mean

Intake Passage Time (IPT), total mass of faeces, percent neutral detergent

fibre (NDF) and percent apparent dry matter digestibility (ADM)

21

Table 4.1 mtDNA control region haplotype frequencies of 55 green turtles at

Gorgona National Park (GNP) in the Colombian Pacific

55

Table 4.2 Composition of green turtle mtDNA haplotypes compared among

nesting, feeding and developmental population data in the Pacific.

56

Table 4.3 Comparison of mtDNA control region sequence diversities in

Gorgona green sea turtle (feeding grounds and other worldwide

populations, measured as haplotype diversity (h) and nucleotide diversity

(π) ± standard error.

57

Table 4.4 Molecular variance (%) of 55 green sea turtles compared among and

between eastern and western Pacific groups of haplotypes identified at

Gorgona National Park.

58

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ABSTRACT

Although the biology and ecology of marine turtles have been widely studied, there

are still knowledge gaps concerning the stock composition, dietary preferences and

nutritional ecology of the green sea turtle (Chelonia mydas) in tropical feeding

grounds of the eastern Pacific Ocean. In this thesis, I incorporate field based

experiments and genetic assays to characterise and describe the foraging ecology and

population composition of green sea turtle juveniles in transitional and developmental

habitats of Gorgona National Park in the Colombian Pacific.

The immature green sea turtle population at Gorgona National Park in the eastern

Pacific showed an omnivorous behaviour, feeding on a range of animal and vegetable

components with a bias towards tunicates (Salps spp.). In contrast to the generally

herbivorous diet of juvenile green turtles (over 40 cm straight carapace length - SCL),

Gorgona’s immature population of large juveniles, sub adults and a few adults fed

mainly on animal matter. Mean SCL ± S.D. of 86 measured turtles was 58.4 ± 7.8 cm

(ranging from 37.0 to 72.9 cm) and mean mass was 28.0 ± 10.7 kg (ranging from 7.5

to 50.5 kg). I speculate that this omnivorous strategy of Gorgona’s immature green

turtles might provide energetic benefits for continuing long distance migrations to

further developmental or mating grounds in the Pacific basin.

To better understand these foraging and nutritional aspects, I investigated the food

digestibility of green turtles in eastern Pacific habitats of Gorgona and calculated the

intake passage time (IPT) of 3 different diets. I collected 150 faecal samples from

turtles (mean SCL of 61.3 ± 4.12 cm and mean mass of 32.3 ± 6.67 kg) to determine

digesta composition and to measure neutral detergent fibre (NDF), acid detergent

iv

fibre (ADF), sulphuric acid lignin and protein. The mean (± S.D.) IPT to recover at

least 73 % of external markers (plastic beads) in the faeces was 23.3 ± 6.6 days. The

true NDF digestibility and dry matter digestibility were determined for high protein

(fish), plant (fresh leaves of Araceae, Moraceae and Bombaceae) and mixed

(combination of both high protein and plant) diets. NDF values obtained for

digestibility of the protein, plant and mixed diets were 1%, 63% and 49%

respectively. There was a large amount of undigested plant material in the faeces,

dominated by fruits of red mangrove (Rhizophora mangle). In this study I considered

the relationships between the type of food, IPT and apparent digestibility in the

context of the nutritional contribution of an omnivorous diet supplied to juvenile

green turtles in Gorgona.

I used mitochondrial DNA control region sequences in order to genetically identify

the stock composition of green sea turtles in developmental and foraging grounds of

Gorgona National Park in the Colombian Pacific. The amplified fragments of the

control region (457 bp) obtained for the 55 turtles aggregation revealed the presence

of 7 haplotypes during that sampling season. The most common haplotype was

CMP4 observed in 84% of individuals, followed by CMP22 (5%). The haplotype (h)

and nucleotide (π) diversities were h = 0.3003 ± 0.0802 and π = 0.008988 ± 0.005049

respectively, showing considerable genetic diversity among and between the groups.

The analyses of phylogenetic inference, using the UPGMA and TCS network methods

confirmed the haplotypes clustered in two geographical regions (north eastern Pacific

and central - western Pacific).

The obtained results suggest for the first time that green sea turtles in the south-

eastern Pacific Ocean combined diets in an opportunistic strategy responding to

v

habitat features and maximum energy acquisition in transitional habitats, as observed

in Isla Gorgona. Moreover, the genetic structure of this mixed juvenile aggregation

demonstrates the diversity of haplotypes from different breeding grounds converging

in foraging grounds of the Colombian Pacific. Based on the study findings, the thesis

highlights the importance of Gorgona as a transitional habitat for conservation

management of green turtles recruited from the central, western and north eastern

Pacific Ocean.

vi

GENERAL DECLARATION In accordance with Monash University Doctorate Regulation 17/Doctor of Philosophy regulations the following declarations are made: I hereby declare that this thesis contains no material which has been accepted for the award of any other degree or diploma at any university or equivalent institution and that, to the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference is made in the text of the thesis. This thesis includes two original papers published in peer reviewed journals and one unpublished publication. The core theme of the thesis is sea turtle nutritional and molecular ecology. The ideas, development and writing up of all the papers in the thesis were the principal responsibility of myself, the candidate, working within the School of Biological Sciences under the supervision of Dr. Richard Reina. The inclusion of co-authors reflects the fact that the work came from active collaboration between researchers and acknowledges input into team-based research. In the case of Chapters 2, 3 and 4 my contribution to the work involved the following: Thesis chapter

Publication title Publication status*

Nature and extent of candidate’s contribution

2 Feeding ecology of the East Pacific green sea turtle Chelonia mydas agassizii at Gorgona National Park, Colombia

Published Conception, execution and writing 90%

3 Intake passage time, digesta composition and digestibility in East Pacific green turtles (Chelonia mydas agassizii) at Gorgona National Park, Colombian Pacific

Published Conception, execution and writing 90%

4 Stock composition of the green sea turtle (Chelonia mydas) in eastern Pacific foraging grounds of Gorgona National Park in Colombia.

in review Conception, execution and writing 90%

Signed: Date: 12th February, 2009

vii

ACKNOWLEDGEMENTS

I want to acknowledge with this thesis the efforts of hundreds of people around the

globe, who are doing what they can to give sea turtles a future. They are my friends,

colleagues and sponsors and I wish to express my gratitude to all of them.

First, I would specially like to thank to my supervisor in Australia, Dr Richard Reina

for his guidance and patience. I thank Richard not only for his friendship and

scientific advice but also for being always suggesting improvements and keeping me

focused on the relevant questions underpinning this study. I want to extend my

gratitude to his family: Lene, Max, Felix, Marjanne and Alf, whom made me feel like

at home during my annual attendance to the Clayton campus. I want to thank

Professor Gordon Sanson who acted as second supervisor until 2004 for his interest in

sea turtle nutrition and his practical suggestions to improve laboratory trials and

nutritional data analysis. My special recognition to the School of Biological Sciences

for supporting my PhD candidature through the faculty of Science Dean’s

Postgraduate Scholarship, making possible the achievement of this personal and

professional goal. My gratitude to my on site supervisor in Colombia, Dr. Fernando

Gast, former Director of the Biodiversity Research Institute Alexander von Humboldt,

for facilitating the use of the Molecular Lab facilities and provide institutional support

for access to genetic resources under Colombian law. Many thanks to Juan D.

Palacio, Carolina Villafañe and Lindamar Mosquera from the Humboldt Molecular

Lab and Cauca University for their advice during the laboratory genetics phase of this

study. My gratitude to the Colombian National Parks Administrative Unit

(UAESPNN) and particularly the Gorgona National Park Staff and kitchen personnel

viii

for their human support and logistical contribution given to this research between

2003 and 2005 fieldwork .

All my gratitude for the volunteers, use of facilities, equipment and materials

provided by the Research Centre for Environmental Management and Development

(CIMAD, Colombia), the International Centre for Tropical Agriculture (CIAT), the

Zoo of Cali (Colombia), the Valle University (Colombia), the University of West

Indies (Barbados), and the Wider Caribbean Sea Turtle Network (WIDECAST). I

also wish to thank the Rufford Small Grants Foundation (UK), the National Fish and

Wildlife Foundation, the Chelonian Research Foundation and Idea Wild from the

United States for funding and field equipment, without their grants and support this

dream have not been possible. I want to acknowledge the warm and friendly

environment on which I was embedded while being at campus by my PhD peers:

Lorenz Frick, Jacquie Salter, Karen Cooper, Sarina Loo, and all those guys that shared

time and fun during my annual visits to Melbourne. Many thanks also to Carol

Logan, who kindly help me to deal with administrative issues at Monash but in

particular for the pleasant chats we had every time she give me a lift between Clayton

and Richard Reina’s house.

Finally, I want to thank my parents, brothers, sisters, friends and all the people who

are working hard to offer a better planet to children like Sabrina, my beloved

daughter, and the ultimate inspiration for carrying out this thesis.

1

CHAPTER 1. GENERAL INTRODUCTION

1.1 Green sea turtles Chelonia mydas agassizii in the eastern Pacific

The green turtle Chelonia mydas is a circumglobal morpho-species made up of

several distinct populations and metapopulations. The common name does not refer

to its external colour, but to the colour of its fat (Hirth, 1997). Several populations of

green turtles have been described based on morphological and genetic characteristics

and some authors propose that Chelonia mydas is comprised of two subspecies:

Chelonia mydas in the Atlantic Ocean and Chelonia mydas japonica in the Indian

Ocean and in the western and central Pacific Ocean (Carr, 1975; Márquez, 1990).

The dark-shelled Chelonia occurring in the eastern Pacific between Baja California

and Peru and western to the Revillagigedos Islands (Mexico) and south to the

Galapagos islands (Ecuador), is recognized by other researchers as a different species,

Chelonia agassizii (Alvarado, Figueroa, 1990; Pritchard, 1999a). However,

mitochondrial DNA genetic data do not support evolutionary distinctness (Bowen, et

al., 1992). The eastern Pacific Chelonia is smaller than the typical Chelonia mydas

and its adult carapace varies from gray to black with brown-green patterns. Juveniles

of green turtles in the eastern Pacific are more colourful and similar to those in the

Atlantic populations (Hirth, 1997). Post hatchlings to 40 cm of straight carapace

length (SCL) are essentially carnivorous during the pelagic stage estimated between 7

months and 5 years (Aguirre, et al., 1994; Carr, et al., 1978). At a size of about 40 cm

SCL young green turtles leave pelagic habitats and enter neritic feeding grounds, at

which time they move to their characteristically herbivorous diet (Bjorndal, Bolten,

1988; Hirth, 1997; Mortimer, 1982) and occupy a feeding niche unique among sea

2

turtles (Bjorndal 1997).

1.2 Feeding and developmental grounds for green turtles

The most important green sea turtle feeding grounds are distributed within the tropics

(Pritchard, 1997), where they have a strong tendency towards herbivory, changing

their diet from carnivorous or omnivorous to herbivorous during their ontogeny

(Bjorndal, 1980; Garnett, et al., 1985; Mortimer, 1982). The recruitment of Chelonia

to neritic feeding and developmental habitats in general occurs at small sizes of 30 -

40 cm SCL (Musick, Limpus, 1997). In several parts of the world adult green turtles

(> 70 - 100 cm SCL) may feed predominantly on sea grasses (Mendonca, 1983;

Mortimer, 1982) or on algae (Bjorndal 1985; Green, 1994; Pritchard, 1971);

depending on their abundance. They will also feed on both types of food when they

are present in the same area (Read, 1991). Variation in diet composition may be a

consequence of local availability of food, turtle selectivity and / or type of habitat

(Bjorndal, 1980; Brand-Gardner, et al., 1999a; Garnett, et al., 1985). The marine

ecosystems of Gorgona National Park in the Colombian Pacific are suitable for the

development of diverse species of algae and marine invertebrates (Bula-Meyer, 1995).

Vegetal and organic material from continental rivers is dragged by convergence of

surface currents into Gorgona’s waters and increases nutrient supply. The insular

near-shore habitats of this protected island 56 km from the continent are considered

the most important habitats for foraging, resting, growth and sexual development of

green sea turtles in the eastern Pacific coast of Colombia (Amorocho, et al., 2001).

Several authors have recognized the importance of insular near-shore habitats for the

development of juveniles (Balazs, 1982; Meylan, et al., 1994) and for adult foraging

(Green, 1994; Limpus, 1994; Seminoff, 2000). Green turtles seem to move through a

3

series of developmental feeding habitats as they grow (Hirth, 1997). It also has been

suggested that developmental and feeding habitats may determine the recruitment

pattern of juveniles and the timing of adult reproductive cycles (Limpus, Reed, 1985).

Consequently, the diet composition and nutritional load of food consumed in Gorgona

is crucial for the development and productivity of green turtles in the eastern Pacific

Ocean.

1.3 Nutritional ecology and importance of transitional habitats for green turtles

Herbivorous reptiles require microbial fermentation in the gut to breakdown plant cell

walls. In consequence, bacteria communities in the digestive tract of green turtles

may affect diet selection. The specificity of the bacterial colony and the capacity to

digest sea grass / algae or animal material, is an important component of the green

turtle foraging strategy (Bjorndal, 1997). Diet quality is determinant for nutrient gain

in the form of Volatile Fatty Acids (VFA) as an important source of energy for green

turtles. Low rate of food ingestion (intake) and high efficient digestibility of

cellulose, hemicellulose and organic matter by gut micro flora determine passage time

and nutrient acquisition in herbivores (Van Soest, 1994). However lignin is the prime

factor influencing the digestibility of plant cell material and as lignin increases the

digestibility, intake and animal performance usually decrease because the amounts of

ADF and NDF increase (Van Soest, Wine, 1967). Low digestibility seems to be

responsible for the low growth rates of green turtles feeding on sea grasses. Under this

consideration, a higher content of protein, or an omnivorous diet should increase

intake passage rate (IPT), influence growth and induce maturity of green turtles

feeding at Gorgona. Nutrient availability is limited by the amount of food and by the

rates of digestive efficiency and ingesta passage through the gut. This can be

4

maximized by ingesting a mixed diet as a way to obtain the essential amino acids

required for protein synthesis and as a mechanism to reduce or eliminate amino acid

wastage while increasing nutrient gain. Thus, we may expect that green turtles at

Gorgona will try to obtain the maximum nutritional benefit by consuming an

omnivorous instead of a completely herbivory diet. This might suppose a digestibility

limitation because of the diverse range of food items they need to eat and the time

required for microbial fermentation to produce VFA. For juvenile green sea turtles in

the eastern Pacific, combined diets are an opportunistic strategy responding to habitat

features and supply to maximize energy acquisition in transitional feeding and

developmental habitats such as the Gorgona Island marine protected area.

1.4 Genetic composition of feeding grounds

Most marine turtles have a life history characterized by a highly dispersive juvenile

stage, marked habitat and diet behaviour shifts through development and long

distance migrations (Bjorndal, 1997; Bjorndal, Bolten, 1988; Carr, et al., 1978;

Godley, et al., 2002). Tag data record and satellite telemetry studies carried out in the

Pacific Ocean have shown a link between developmental habitats and specific adult

foraging habitats, as well as with specific nesting beaches (Balazs, 1982; Seminoff, et

al., 2008). In addition, mitochondrial DNA polymorphisms have proven useful to

identify the origin of marine turtles in migratory corridors or at coastal feeding

grounds (Bowen, 1995). Today, molecular genetic markers are combined with

tagging data to identify green turtle stocks and track animals along migratory

pathways from nesting to feeding grounds (Dethmers, et al., 2006; Dutton, et al.,

2008). The shallow waters of Gorgona National Park are feeding habitats that harbor

aggregations of immature green turtles with different carapace shapes and colours.

5

Morphologic characteristics may thus correspond to distinct haplotypes and they can

be attributed to genetic mixture in a feeding ground with contributions of juveniles

recruited to Gorgona from the eastern, central and western Pacific. Elucidating

population structure and dispersal patterns of green sea turtles in the eastern Pacific

will be useful for conservation management of this endangered species through the

entire basin. Multinational efforts are required to protect individuals at foraging

grounds beyond the boundaries of the state nation from where they originate.

Protecting juveniles foraging and developing along neritic habitats of Gorgona thus

implies the protection of rookeries thousands of kilometres away from this Colombian

island.

1.5 Aims of the study

The aims of this study were to combine ecological, nutritional and genetic approaches

to characterize the green sea turtle population in eastern Pacific neritic habitats of

Gorgona National Park in Colombia, South America. I investigated the class-sizes of

the green turtle population occurring in coral reefs and waters of this marine protected

area, as well as their dietary preferences in order to identify the foraging behaviour

and contribution of consumed food items in terms of energy and productivity. The

nutritional supply was measured through neutral detergent fibre (NDF) and acid

detergent fibre (ADF) methods; protein and Organic Matter analyses, carried out to

revealed the relationship between IPT, digestibility and energy acquisition by semi

captive turtles under animal, vegetal and omnivorous diets. I also determined the

stock composition of juveniles converging at this island and the presence of identified

haplotypes from varied and distant origins, to investigate the importance of stopover

refuelling stations like Isla Gorgona for maintaining gene flow. I considered the

6

implications of these results for conservation management of green sea turtle

populations in the Pacific Ocean.

1.6 Thesis structure

This thesis is made up of 5 chapters including this introductory chapter. Chapter 2

is a published paper (Amorocho & Reina, 2007) on the description of the green sea

turtle, Chelonia mydas, feeding ecology on the eastern Pacific habitats of Gorgona

National Park in Colombia. Chapter 3 is a published paper (Amorocho & Reina,

2008) on the nutritional aspects of three different diets administered to semi captive

turtles considering the relationships between the type of food, IPT and apparent

digestibility in the context of the nutritional contribution of an omnivorous diet. The

genetic composition of the feeding stock and the geographical origin of haplotypes

identified on juveniles recruited to Gorgona are presented in Chapter 4. These 3

chapters have been written in journal format incorporating abstract, introduction,

methods, results, discussion and acknowledgements, as they were submitted for peer

review before being incorporated into this thesis. I provide a combined reference list

for all chapters at the end of the thesis. In Chapter 5, the findings of the different

components of the research are synthesised and discussed in the context of the

nutritional contribution of food ingested by immature green turtle aggregation

composed of individuals from different Pacific Ocean sites converging in Gorgona

Island.

7

CHAPTER 2. FEEDING ECOLOGY OF THE

EASTERN PACIFIC GREEN SEA TURTLE

CHELONIA MYDAS AT GORGONA NATIONAL

PARK, COLOMBIA.

Declaration for Thesis Chapter 2

In the case of Chapter 2, the nature and extent of my contribution to the work was the

following:

Nature of contribution Extent of contribution (%)

Conception, execution and writing 90%

The following co-authors contributed to the work.

Name Nature of contribution Extent of contribution (%) for student co-authors only

Richard D. Reina

Advice, interpretation and writing assistance

Candidate’s Signature

Date 12/2/09

Declaration by co-authors

The undersigned hereby certify that:

(1) the above declaration correctly reflects the nature and extent of the candidate’s

contribution to this work, and the nature of the contribution of each of the co-

authors.

(2) they meet the criteria for authorship in that they have participated in the

conception, execution, or interpretation, of at least that part of the publication in

their field of expertise;

8

(3) they take public responsibility for their part of the publication, except for the

responsible author who accepts overall responsibility for the publication;

(4) there are no other authors of the publication according to these criteria;

(5) potential conflicts of interest have been disclosed to (a) granting bodies, (b) the

editor or publisher of journals or other publications, and (c) the head of the

responsible academic unit; and

(6) the original data are stored at the following location(s) and will be held for at least

five years from the date indicated below:

Location(s) School of Biological Sciences, Monash University, Clayton

Campus

Signature 1

Date 12/2/09

15

CHAPTER 3. INTAKE PASSAGE TIME,

DIGESTA COMPOSITION AND DIGESTIBILITY

IN EAST PACIFIC GREEN TURTLES (CHELONIA

MYDAS AGASSIZII) AT GORGONA NATIONAL

PARK, COLOMBIAN PACIFIC.



following:





Richard D. Reina



Date 12/2/09





authors.

16













Campus

Signature 1

Date 12/2/09

Author's personal copy

Intake passage time, digesta composition and digestibility in East Pacific green turtles(Chelonia mydas agassizii) at Gorgona National Park, Colombian Pacific

Diego F. Amorocho, Richard D. Reina ⁎School of Biological Sciences. Monash University, Clayton, Victoria 3800, Australia

A B S T R A C TA R T I C L E I N F O

Article history:Received 6 February 2008Received in revised form 25 February 2008Accepted 9 April 2008

Keywords:DigestibilityIntake passage timeGreen turtleNutrition

We investigated the food digestibility of East Pacific green turtles, Chelonia mydas agassizii, at tropical coralreefs of Gorgona National Park in the Colombian Pacific and calculated the intake passage time (IPT) of 3different diets. We collected 150 faecal samples from turtles (mean straight carapace length 61.3±4.12 cmand mean mass 32.3±6.67 kg) to determine digesta composition and for measurement of neutral detergentfibre (NDF), acid detergent fibre (ADF), sulphuric acid lignin and protein. The mean (± S.D) IPT to recover atleast 73% of external markers (plastic beads) in the faeces was 23.3±6.6 days. The true NDF digestibility anddry matter digestibility were determined for high protein (fish), plant (fresh leaves of Araceae, Moraceae andBombaceae) and mixed (combination of both high protein and plant) diets. NDF values obtained fordigestibility of the protein, plant and mixed diets were 1%, 63% and 49% respectively. There was a largeamount of undigested plant material in the faeces, dominated by fruits of red mangrove (Rhizophora mangle).We considered the relationships between the type of food, IPT and apparent digestibility in the context of thenutritional contribution of an omnivorous diet. Our results suggest that for juvenile green sea turtles in theEastern Pacific combined diets are an opportunistic strategy responding to habitat features and supply tomaximise energy acquisition in transitional habitats such as Isla Gorgona.

Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.

1. Introduction

The acquisition of nutrients for all animals depends not only onfeeding but also on mechanisms of digestion and absorption toprocess the foods obtained in an interrelated set of needs andprocesses (Will et al., 2004). The green sea turtle (Chelonia mydas) isconsidered to be primarily a herbivore that feeds throughout most ofits range on seagrass pastures, but that will also consume some animalmatter (Seminoff et al., 2006), which it readily accepts in captivity(Mortimer,1982). Green turtles can feed on algaewhere seagrasses arenot available (Bjorndal, 1980; Bjorndal 1985; Forbes, 1994; Read, 1991)or consume both when they are present in the same area (Brand-Gardner et al., 1999; Ferrerira et al., 2006). Other plant material suchasmangrove leaves and fruits have been described as a substantial andnutritionally important part of green turtle diets in Australia (Limpusand Limpus, 2000) and juveniles of the East Pacific green turtle(Chelonia mydas agassizii) in Colombia (Amorocho and Reina, 2007).Consumption and effective digestion of a plant diet by sea turtlespresents a significant challenge. Turtles rely on microbial fermenta-tion in the large intestine to digest plant cell walls, but they can notchew their food to reduce particle size and facilitate this process(Bjorndal et al., 1990) because they don't have teeth. Thus, only by

retaining material for longer in the gut with a slow passage rate canthey equal ruminants in the digestion of plant material by fermenta-tion. The primary nutritional end products of this fermentation are thevolatile fatty acids (VFA), an important source of energy in herbivoroussea turtles (Bjorndal, 1997). The quality of forage is a major deter-minant of the efficiency of digestion and can be assessed by mea-surement of acid detergent fibre, (ADF, cellulose and lignin) andneutral detergent fibre (NDF, cellulose, hemicellulose and lignin) usingthe detergent system (Van Soest, 1963; Van Soest and Wine, 1967).Lignin is the prime factor influencing the digestibility of plant cell wallmaterial and as lignin increases the digestibility, intake and animalperformance usually decrease because the percent ADF and NDFincrease (Van Soest, 1994).

Green sea turtle juveniles over about 40 cm straight carapacelength (SCL) shift their feeding behaviour from carnivorous toherbivorous when they move from pelagic to neritic developmentalhabitats (Bjorndal and Bolten, 1988; Hirth, 1997; Musick and Limpus,1997). In turtles that feed on seagrass, Thalassia testudinum, in theBahamas, nutritional assimilation occurs at a very low intake rate withhigh efficiency digestibility of cellulose (89%), hemicellulose (75%) andorganic matter (67%) by cellulolytic microflora present in the gut(Bjorndal, 1980). The same author suggests that the digestibilitycoefficients of NDF and ADF of green turtles foraging selectively onyoung blades of the seagrass reflect the specificity of their intestinalmicroflora. Bjorndal (1997), proposed that the optimal forage forgreen turtles may be that to which its gut microflora are adapted

Journal of Experimental Marine Biology and Ecology 360 (2008) 117-124

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(either seagrass or algae). Thus, it is expected that a less selective dietor one which is mixed (omnivorous) may change the composition andfunction of fermenting bacteria to affect the efficiency of digestion.However, nutrient gain in the form of VFA may be maximised by theability to ingest a greater quantity of food more rapidly (Bjorndal,1997).

Large juvenile and adult East Pacific green sea turtles are describedas primarily herbivorous feeding on seagrasses and/or marine algae inGalapagos and Mexico (Fritts, 1981; Green, 1983; Seminoff et al.,2002). Nonetheless, along the Coast of Baja California, they occasion-ally consume red crabs (López-Mendilaharsu et al., 2005) and otherchordates (Casas-Andreu and Gómez-Aguirre, 1980). A significantamount of animal matter (molluscs, amphipods, sardines andanchovies) was found in the stomachs of subadult and adult greenturtles captured in the Pacific coastal waters of Peru (Hays and Brown,1982) and fish eggs, molluscs, polychaetes and jellyfish have beenrecovered from the stomachs of turtles caught near the Pacific coast ofEcuador (Fritts, 1981). These findings suggest a carnivorous trend inthe feeding habits of East Pacific green turtles in the region. Thisbehaviour has also been observed in individuals consuming a mixeddiet of tunicates (Salpas spp.), terrestrial leaves (Ficus spp.) and algae(Gelidium spp.) at Gorgona National Park, in the Colombian Pacific(Amorocho and Reina, 2007). The high frequency of tunicates (73.8%)recovered through oesophageal lavages conducted in 84 juveniles(N40 cm SCL), showed that Colombian green turtles are omnivoreswith a diet biased towards animal matter (Amorocho and Reina,2007). Sea turtles consistently ingesting a mixed diet would almostcertainly develop a different microbial community than exclusivelyherbivorous turtles, in order to degrade the various complexcarbohydrates required to digest each food item efficiently (Bjorndal1985). So, long intake passage time (IPT) might occur to enable highlyefficient fibre digestibility because the microbial populations in thegreen turtle gut may have to adapt continually to changes in the typeand proportions of selected diet components.

Other than stomach content analyses and anatomical descriptions(Bjorndal, 1997; Green, 1994; Green and Ortiz, 1982; Hays and Brown,1982), studies on the digestive system of green turtles in the southeastPacific are lacking. Information about the digestibility of a mixed diet

will indicate the real benefit to turtles of eating animal matter toincrease energy gain in a transitional habitat such as Gorgona.Nutrient availability is limited by the amount of food and bycompetition between the rates of digestion and passage. This can bemaximised by ingesting a mixed diet as a way to obtain the essentialaminoacids required for protein synthesis and we may expect thatgreen turtles at Gorgona will try to obtain the maximum nutritionalbenefit by consuming an omnivorous diet. This might suppose adigestibility limitation because of the diverse range of food items theyneed to eat and the time required for microbial fermentation toproduce VFA. To elucidate these nutritional aspects of the East Pacificgreen turtle population foraging at Gorgona National Park in theColombian Pacific, we investigated the apparent digestibility of threetypes of diet, measured IPT and considered the effect of diet quality onjuveniles' growth. We addressed the following questions: 1. Do turtlesingesting an omnivorous diet in Gorgona have low digestiveefficiencies? 2. If so, can nutrient gain be maximised by the abilityto ingest a greater quantity of different types of food more rapidly? 3.If the East Pacific green turtle has a slow fermenting strategywith highdigestibility of fibre and organic matter, is ingesta retained within thefermentation chamber for long periods? By revealing these aspects ofdiet selection and digestive process, we can begin to understand hownutrition acts as a regulating mechanism for growth and maturity injuveniles and the importance of Gorgona as a foraging and develop-mental habitat for the East Pacific green sea turtle.

2. Materials and methods

2.1. Study site

Fieldwork was carried out at Isla Gorgona National Park (2° 50' - 3°00'N, 78°10' - 78°15' W) in Colombia. The island is 9 km long and2.5 km wide with a total protected area of 617 km2 and a maximumheight of 338 m, located 56 km offshore from the town of Guapi in thesouthern Colombian Pacific coast (Fig. 1). The shores of Gorgona arepredominantly steep plunging cliffs, with small sandy and pebblybeaches supplied on its eastern side by coral reef detritus. The island issurrounded by near-shore coral reefs where East Pacific green sea

Fig. 1. Map of Gorgona National Park in the Colombian Pacific, showing location of the sea turtle in-water enclosure.

118 D.F. Amorocho, R.D. Reina / Journal of Experimental Marine Biology and Ecology 360 (2008) 117-124


turtles can be found resting or foraging. The IPT, digesta compositionand digestibility experiments were conducted within the constraintsof limited logistic and infrastructure conditions in the field. Gorgona isan isolated place without permanent electricity to adequately storecollected samples for some specialised analyses.

2.2. Turtles sampled, confinement and faecal collection

We conducted IPT and digestibility experiments between July andOctober 2005. Nine green turtles were caught by hand using snorkelat a depth of up to 7 m in coral reefs of La Azufrada and Playa Blanca.Straight carapace length (SCL) of all animals was measured asdescribed by Bolten (1999) and turtles were weighed and doubletagged in the front flippers with Inconel 1005-681S tags (NationalBand & Tag Co.), following standard techniques (Balazs, 1999). Aplastic Ziploc bag cut at the bottom was wrapped inside anotherplastic bag and covered with nylon cloth to be attached to theanimal's cloaca using a surgical thread, leaving the zip side at the endto enable collection of faeces in a modification of Bjorndal'stechnique (1980). Turtles were confined in an enclosure made up ofbamboo, PVC and plastic mesh, in water of approximately 3 m depth(Fig. 2). Three turtles were held at a time, each in a 2 m×2 m×2 mseparated compartment of the in-water enclosure for 3 consecutivesampling periods. Turtles were acclimated to the enclosure for 7 daysand fed with fresh leaves and small fish supplied ad libitum. Wecovered the enclosure with 6 mm square plastic mesh to reduce thepossibility of turtles eating food other than that provided in thelaboratory and we checked the enclosure daily to keep it free ofleaves, small fish and tunicates.

2.3. IPT and dietary treatments

After acclimation, we administered external markers to each turtlebefore being feeding it with the experimental diet and placing it backinto the enclosure. External markers are synthetic indigestible organicsubstances such as plastic beads, that are used in gut passage studiesbecause they can be counted, are not chewed into smaller pieces andcan easily be recovered from the faeces (Van Soest, 1994). This isprobably the best single measure of food passage through the entiregut if faeces can be collected (Warner, 1981). We used cylindricalyellow beads (2.0 - 3.0 mm×1 mm packaged into gelatin capsulescontaining 20 beads each) and introduced between three and fivecapsules into the lower oesophagus by pushing them through a plastichose. IPT was calculated from the integrated average of markersrecovered in the faeces following administration.

Animals were moved every three days into the laboratory to be fedwith 1 – 2% body mass of wet food as a maintenance diet (Higgins,

2003). The three experimental diets given to the turtles were: highprotein (fish); plant (a blend of Araceae, Moraceae and Bombacaeaeleaves) andmixed (a combination of animal and plant material). Mealswere prepared in the form of approximately 8 cm3

fish cubes and smallpackages of fresh leaves. The mass of food given each time varied withthe willingness of the turtle to ingest it. The diet rations were intro-duced using 2 plastic hoses to push the food deep into the oesophagus.The high protein group was composed of turtles with mass rangingfrom 27 to 31 kg; the plant group mass was from 31 to 36 kg and themixed diet group was from 33 to 48 kg. The three dietary treatmentsand mass of food administered to animals are presented in Table 1.

Turtles were taken out of the water every day at 3 p.m. and faecescollected from each individual's attached bag before returning them tothe enclosure. Mean value for water temperature recorded throughoutthe three experimental seasons at 3 m below the surface, was 28.3±0.3 °C; ranging from a minimum of 27.7 °C to a maximum of 29.0 °C.We measured body mass every 7 days and replaced faecal collectionbags as required.

2.4. Digesta composition and digestibility analyses

Collected faecal material was initially sorted into categories (fruits,leaves,mixture, coral and plastic).Wetmasswasmeasured for thefirst 3categories and then samples were preserved in 70% ethanol in plasticbags and stored at 4 °C in a portable fridge for organicmatter (OM), aciddetergent fibre (ADF), neutral detergent fibre (NDF) and lignin analyses.At the conclusion of the experiment, attached cloacal bags werecarefully removed from turtles and iodine (10%) applied around thecloaca before releasing the animals at the approximate site of capture.

Faecal samples were dried to constant mass at 60 °C for 48 to 60 hand stored in desiccators (Wood andWood, 1981). Following return tothe mainland, samples were dried again at 105 °C for 12 hours,

Table 1Total mass (g) of protein and plant feeding rations supplied every 3 days to 9 turtlesconfined in a near shore captive enclosure at Gorgona National Park, Colombia

Turtle Type of diet Plant ration (g) Protein ration (g) Total intake (g)

T1 Mixed 175 320 495T2 Mixed 225 100 325T3 Mixed 125 85 210T4 Mixed 223 210 433T5 Mixed 500 500 1000T6 Plant 292 - 292T7 Plant 300 - 300T8 Protein - 534 534T9 Protein - 500 500

Fig. 2. In-water enclosure to keep turtles confined near shore El Poblado beach. The enclosure was sunk to the water level after being placed in position.

119D.F. Amorocho, R.D. Reina / Journal of Experimental Marine Biology and Ecology 360 (2008) 117-124


weighed and ground in a Wiley Mill. Coral and plastic debris wereexcluded because they were not digested and were probablyconsumed incidentally by turtles while feeding on other food items.Faecal samples were aggregated for the whole collection period ofeach animal, then all faecal and food samples were analysed fororganic matter by heating in a muffle furnace at 550 °C for 12 h. Totalnitrogen was measured by the micro-Kjeldhal method and crudeprotein including both true protein and non-protein nitrogen wasestimated by multiplying total nitrogen content by 6.25, while ADF,NDF and sulphuric acid lignin analyses were performed followingstandard procedures (Van Soest, 1963; Van Soest and Wine, 1967).Food samples were also analysed for organic matter, crude protein,ADF, NDF and lignin. Hemicellulose content was calculated bysubtracting ash-free ADF from ash-free NDF, and cellulose wascalculated by subtracting lignin from ash-free ADF following proce-dures applied for green turtles previously (Bjorndal, 1980). Duplicateanalyses were performed when the quantity of material permitted.

3. Results

All results are reported as the mean±S.D.

3.1. Animal size and gut length

The mean SCL of the 9 studied turtles was 58.2±3.6 cm. (rangingfrom 52.2 to 62.2 cm) and mean mass was 32.3±6.7 kg (ranging from26 to 48 kg). One of the turtles (T7) died 12 days after capture and anecropsy revealed a 5 cm long-line “J” shaped hook # 7 (Mustard Co.)embedded in the oesophagus, that was presumably accidentallyingested by the turtle from some fisheries activity prior to capture.Large pieces of plastic debris were distributed all along the gut andmay have also contributed to the cause of death. The entire digestivetract of this turtle (63.7 cm SCL, 36.0 kg mass) from the beak to the

cloaca was 6.53 m in length. The necropsy showed that plant matterconsumed by the turtle had travelled along the gut in the form ofingesta packages (bolus), with fresh leaves observed between theoesophagus and the stomach (0 - 70 cm), boluses of partially digestedplant material located in the midgut (70 - 318 cm) and large amountsof mangrove fruits were distributed from mid to hindgut (318 -650 cm). All of the external markers given to the animal on day one ofthe experiment were contained within boluses distributed along themidgut (371 - 411 cm) at the time of death 12 days later.


No markers were recovered from Turtle T1 after 38 days and onlyone marker was recovered from turtle T2 in 32 days, so these twoturtles were released to avoid health problems that could result fromextended captivity. Excluding T1, T7 and T8 due to zero or low markrecovery, the mean IPT for the 6 remaining turtles was 23.3±6.6 days(559 hr) ranging from 22.0±6.9 days (528 hr) for the first recovery dayand 24.7±6.6 days (593 hr) for the last recovery day of markers(Table 2). We ended the experimentwhenwe recovered at least 73% ofbeads from faeces (Fig. 3).

3.3. Digesta composition and digestibility analyses

Faecal sample percentage wet mass of mangrove fruits, leaves,remains composed of fragments of leaves and metabolic excretionstermed ‘mixture’, coral and plastic debris are shown in Fig. 4. Entire or

Table 2Recovery of administered markers from 6 turtles. Mean Ingesta Passage Time (IPT)=23.3±6.6 days

Turtle SCL(cm)

Number ofadministeredmarkers

Percentageof markersrecovered

First recoveryday

Last recoveryday

T2 59.0 100 96% 16 17T3 60.5 95 86% 14 19T4 60.8 60 100% 19 21T5 54.8 60 82% 23 27T6 62.2 60 100% 30 32T9 52.0 64 73% 30 32Mean±SD 58.2±3.6 73.2±17.3 89.4±10.0% 22.0±6.3 24.7±6.0

T1 was excluded because no markers were recovered by day 38 and animal wasreleased. T7 died after 12 days of treatment due to a hook ingested prior to experiment.T8 was released after day 31 without releasing markers.

Fig. 3. Percentage of markers recovered and time elapsed for over 73% of markers to berecovered from faecal samples of 6 green turtles under 3 dietary treatments. P=plant,M=mixed, F=fish (high protein).

Fig. 4. Percentage of different categories of ingested items collected from faecal samplesof 9 green sea turtles at Gorgona National Park.

Table 3Percentages of organic matter (OM), neutral detergent fibre (NDF), acid detergent fibre(ADF), lignin and protein of diet components and 3main faecal categories (leaves, fruits,mixture) collected from 9 green turtles at Gorgona National Park, Colombia

Diet Sample OM(%) NDF(%) ADF(%) Lignin(%) Protein(%)

Fish 87 1 0.1 0 98Leaves 73 63 53 38 8

Faecal Sample OM(%) NDF(%) ADF(%) Lignin(%) Protein(%)

MIXTUREMean±SD 68±3 74±19 58±6 28±5 16±2Min 63 0 47 20 12Max 74 87 68 36 19

LEAVESMean±SD 52±33 45±42 45±27 22±13 12±8Min 3 0 6 5 2Max 74 87 68 36 19

FRUITSMean±SD 58±24 61±30 49±20 25±11 14±6Min 3 0 6 5 2Max 74 87 68 36 19



chopped pieces of mangrove fruit were present in faeces throughoutthe experiment in all 9 assessed turtles. Percentages of OM, NDF, ADF,lignin and protein from fish and plant dietary treatments, as well asthe 3main faecal components are presented in Table 3. Through directobservation of the faecal material it appears that animal food (i.e. fish)was entirely digested, but some plant material (particularly mangrovefruits) showed little visible sign of digestion (Fig. 5). True NDF andapparent dry matter digestibility (ADM) of the assessed turtles arepresented in Table 4.

4. Discussion

4.1. Turtle size class

East Pacific green turtles using coral reefs and surrounding watersof Gorgona National Park were small and large juveniles (52.0 – 62.2

SCL), confirming recruitment of young turtles at the size of 50.0 -70 cm SCL to neritic habitats of this protected area. The mean size of58.2±3.6 cm at Gorgona is similar to that measured from juveniles atBahía Magdalena and Estero Banderitas in Baja California Sur, Mexico(López-Mendilaharsu et al., 2005) and is typical of juveniles on thePacific coast of the Baja California Peninsula (Koch et al., 2007) inprotected reefs or mangrove habitats where they can find food andshelter. However, juveniles in Gorgona, consume a mixed diet biasedtowards animal matter (Amorocho and Reina, 2007) while those inBahía Magdalena feed mostly on algae and seagrasses (López-Mendilaharsu et al., 2003; López-Mendilaharsu et al., 2005; Seminoffet al., 2002). At Gorgona, it is advantageous for juvenile green turtlesto consume a high protein diet with good nutrient assimilation tocontribute energy for growth while juveniles migrate betweenforaging and developmental habitats. In comparison, stable isotopesin scutes of N36 cm young green turtle recruited to neritic habitats offGreat Inagua (Bahamas), reveal rapid ontogenetic shift from carnivor-ous to herbivorous lifestyle (Reich et al., 2007). Green sea turtlejuveniles at a size class of 50 - 70 cm continue feeding mainly onanimal matter or at least consuming an omnivorous diet in neriticnear shore habitats of Gorgona (Amorocho and Reina, 2007) in anontogenetic pattern different to that observed in green turtles of theCaribbean. These different dietary patterns are probably indicative ofthe transitional nature of the Gorgona habitat as a stop-over point inturtle migration.


There is very little information about the use of external markers(beads) to estimate time elapsed between ingestion and egestion insea turtles. The percentage of bead recovery in faeces of 6 turtles wasnot always over the 95% typically applied in ruminant passage studies(Van Soest, 1994). However, our mean of 89% (from the 73% to 100%range) of markers recovered was similar to the 88% - 100% collected in3 juveniles of similar size class (50.3 – 55.2 cm SCL) in Australia (Brandet al., 1999). It is possible that some markers were lost through smallholes between the collection bags and the skin around the cloaca. It isunclear why no markers were recovered after at least 32 days from 2turtles. We suspect that they may have been regurgitated followingadministration and escaped through the mesh of the enclosure butcannot exclude the possibility that their movement through the gutwas retarded for some unknown reason.

Mean IPT for captive turtles was long with a mean time of first andlast marker recovery 528 to 593 h compared to digesta retention timesof 156 to 325 h estimated in wild immature green turtles at Moreton

Fig. 5. Pieces of a) the mangrove fruit Rhizophora mangle and b) leaves of Ficus spp.collected in the faeces of 9 assessed turtles.

Table 4Number of days that turtles were confined, total food intake, mean Intake Passage Time(IPT), total mass of faeces, percent neutral detergent fibre (NDF) and percent apparentdrymatter digestibility (ADM) of 9 green sea turtles at Gorgona National Park, Colombia

Turtle Daysconfined

Numberof feeds

Totalintake (g)

IPT(days)⁎

Wet massof collectedfaeces (g)

NDF(%)

ADM(%)

T1 38 13 3519 - 725 77 89T2 25 8 5850 16 866 82 67T3 19 6 1853 16 321 77 75T4 25 9 4138 25 841 75 78T5 31 10 10000 - 3377 60 66T6 19 7 2051 20 687 62 67T7 13 4 1200 - 1649 -61 -37T8 31 11 5674 31 535 19 91T9 31 10 5000 31 746 22 85Mean±SD 26±8 9±3 4365±2700 23.3±7.2 1083±933 59±25 77±10

⁎ Mean time elapsed between first and last recovery day of markers . NDF and ADMvalues of T 7 are negative because intake was less than the excreta produced after12 days of captivity prior to death from unrelated hook ingestion. These values were notincluded for mean and SD estimation.



Bay, Australia (Brand et al., 1999). This difference is a consequence ofthe experimental design and different techniques used in both studies.In the Moreton Bay study, turtles were freed into the wild to resumefeeding after the markers were administered, then recaptured andsacrificed after 5 – 9 days. This was before they excreted any beads, soIPT was estimated from beads counted at some point along the gutfollowing necropsy. We think that our measurements of IPT are moreprecise because the passage of beads was measured all the waythrough from ingestion to egestion, with excreted beads collecteddaily in attached cloacal bags. Additionally, the necropsy of turtle T7that died from the unrelated hook ingestion, showed that markerswere only half to two thirds the way along the gut after nearly 300 h(12 days). The point of the gut that the markers reached was similar tothat of Brand et al. (1999) in a comparable time, but our resultsindicate that it took about another 11 days for markers to reach theend of the gut and be excreted.

4.3. Digesta composition

The presence of large amounts of undigested mangrove fruits infaeces of all sampled turtles reflects the abundance of this food itemin Gorgona waters. Mangrove fruits are found drifting in surfacecurrents carrying organic matter from the mainland rivers of la Tola,Sanquianga and Patia in South Western Colombian Pacific. Theabundance and availability of Rhizophora mangle fruits in superficialwaters of Gorgona varies between seasons and may influence die-tary changes in foraging green turtles from plant to animal matter(tunicates). The presence of undigested mangrove fruits in the faecesof all turtles can be associated with seasonal abundance prior to theexperiment. The low percentage of NDF (63%) in freshmangrove fruitsmight be responsible for the high intake of this food item before ourstudy was conducted. The consequence of consuming Rhizophoramangle needs to be considered in further studies, since mangrovesplay a key role in the nutritional ecology of immature green turtlepopulations in the Pacific (Limpus and Limpus, 2000).

4.4. Digestibility

Fromdirect observation of digesta composition in the entire gut, it isevident that green turtles at Gorgona are opportunistic consumers.Distinct packages of food separated in boluses containing mangrovestalks, small fish and leaves reflects the omnivorous behaviour ofjuveniles in thewild. This also indicates how often they switch betweenanimal and plant food items. A similar pattern of shifting diet wasobserved in Australia, with separate boluses representing differentfeeding bouts divided along the digestive tract (Brand et al., 1999).

The NDF digestibility of six turtles from which we recoveredmarkers varied, with apparent dry matter digestibility (ADM) highestin the protein (fish) diet group, lowest in the plant diet group andintermediate in the mixed diet group, although sample size was smallin the first two groups. The protein diet (NDF 21±2%) seems to be welldigested, with our measured ADM of 85 – 91% very similar to that forgreen turtles (82 – 90%) fed commercially prepared high protein diets(Hadjichristophorou and Grove, 1983; Wood and Wood, 1981). As aresult, juveniles in Gorgona might grow more rapidly than thosefeeding on sea grass or algae in the Caribbean (Bjorndal, 1985) andAustralia (Brand et al., 1999), considering protein as the limitingnutrient responsible for growth and sexual maturity. Thus, we concurwith Bjorndal (1985) that growth rates in wild green turtles are undernutritional rather than genetic control. We propose that the need forrapid growth is indicated by the diet biased toward animal matter ofjuvenile turtles at Gorgona (Amorocho and Reina, 2007), in contrast tothe assumption that juveniles must exclusively consume plant matteras they approach adulthood.

For animals fed with plant diet, 63% of organic matter consisted ofNDF and ADM was 67%, in agreement with the ADM of 66% measured

from green juveniles consuming Thalassia testudinum blades fromgrazed plots in Union Creek, Bahamas (Bjorndal, 1980). NDF digest-ibility for turtles varies depending on its composition, becausehemicellulose can be partially digested by sea turtles but lignin cannot. In addition, lignin and cutin appear to reduce digestibility of otherNDF components. NDF digestibility also depends on the species and itsdigestive anatomy, size and strategy and so ruminants digest moreNDF than hind-gut fermenters such as sea turtles.

4.5. Comparisons with ruminants

In general there are two options available for a herbivore: to eithermaximise digestive efficiency or to maximise intake (White et al.,2007). These are competing process because digestion is a time-dependent process, so that maximum digestion requires a long time. Ifeach unit of forage must be held in the digestive tract a long timeduring microbial fermentation, then a species maximising digestiveefficiency cannot eat a large amount of food because the capacity ofthe gut is limiting. Conversely, a species maximising intake willsacrifice digestive efficiency because rate of passagemust be increasedto make room for the additional forage and therefore potentiallydigestible material will pass through the digestive tract (Mertens andEly, 1979). Ruminants are more efficient at digesting the low andmedium quality forage normally consumed by these animals, usuallyin 24 - 48 h, but ruminants are constrained by rumen fill and rate ofpassage on how much they can eat in a day (Leng, 1990). Hind-gutfermenters like green sea turtles are not similarly constrained andthus can compensate for lower digestive efficiency by simply eatingmore. When food is not limiting, hind-gut fermenters can attain anequal or higher rate of energy (or digestible dry matter) intake.Ruminants may be at an advantage when food quantity is limited, buthind-gut fermenters such as green sea turtles usually have the optionof consuming more of a higher quality food, which is generallyabundant in Gorgona. Analysis of plant and animal characteristics thatinfluence digestion and intake suggests that maximum intake ofdigestible drymatter is influencedmore by the proportion of fiber thatis indigestible and the rate of passage than by the rate of fiberdigestion. Thus, with a slow passage rate and increased digestibility,the green turtles at Gorgona maximise nutrient gain in the form ofVFA through an omnivorous diet.

4.6. Temperature implications

Temperature can be a major variable limiting digestive efficienciesin poikilothermic animals. For this reason, the intake rate anddigestive efficiency of turtles will be affected by geographical andseasonal variations in water temperature. Body temperature of greenturtles in Gorgona are not more than one or two degrees C differentto the water temperature at 3 m depth, which is not sufficient tosignificantly affect IPT or decrease digestibility. Thus, the microbialincubation temperature of these turtles was considerably cooler thanthat of endothermic ruminants, further contributing to relativelylengthy gut passage times. Reptiles can attain digestive efficienciesroughly comparable to those of ruminants by subjecting feed tomicrobial fermentation processes for longer periods of time (Mackie,2002). This strategy is not feasible for endothermic herbivores with arapid metabolic rate and limited time available to extract energy andnutrients (Farlow, 1987). Thus the main advantage of mastication inruminants is to reduce the time needed to attain a digestibility thatreptiles can achieve simply through a longer IPT. Consumption of amixed diet and a regulated temperature are the key aspects to main-tain the efficiency of themicrobial fermentation process driving intakerate, passage time and digestibility of green turtles in developmentaland feeding grounds of Gorgona. Water temperatures of 21.3 - 25.5 °Crecorded in feeding grounds of Moreton Bay, Australia (Brand et al.,1999) and of 19 – 28 °C in Baja California, Mexico (Lluch-Belda et al.,



2000), are similar to those measured in Gorgona and are typical oftropical waters. Fluctuation in temperature over the course of the yearwill have some effect on the fermentation process, digestive efficiencyand IPT for a poikilotherm animal. This might be the case for turtlesfeeding on seagrasses in the Bahamas during atypical El Niño years,when normal temperature ranging from 24 – 29 °C rises to above 34 °C(Bjorndal, 1980), increasing the rate of digestion and food intake.

4.7. Conservation and management issues

The death of turtle T7 due to a hook embedded in its oesophagusraises the issue of high sea turtle mortality in artisanal and industrialfisheries. Incidental capture in long-line fishing gear has beendocumented as the major threat for green sea turtle populations inthe Pacific Ocean (Seminoff, 2004). Technological changes in currentfishing practices and the use of de-hookers need to be rapidlyintroduced in order to reduce green sea turtle by-catch in Colombia. Inaddition, the amount of plastic (1.6%) that we recovered from thefaeces of turtles indicates the magnitude of marine pollution affectingthe feeding behaviour and health of green sea turtles. It is important tocontinue documenting the occurrence and impact of plastic debris indiets consumed by sea turtles for the design and implementation of aregional management strategy in the Pacific. Results from our studyshow how the transitional feeding grounds of Gorgona National Parkprovide nutritional opportunities that benefit turtles' digestiveefficiency that can be translated into growth and fitness. For managersto better protect them, it would be important to address theabundance of debris and quality of food items ingested by omnivorousjuveniles in the protected habitats and to ensure that Gorgonaremains available as a transitional habitat.

4.8. Conclusions

It seems that for juvenile green sea turtles in the Eastern Pacificcombined diets are an opportunistic strategy responding more tohabitat features and supply rather than to the species' herbivorouspreference. Perhaps foraging patterns in juveniles are driven moreby the rookery geographical distribution, type of habitat and foodavailability than by ontogenetic changes experienced by animalswhen they enter neritic shallow waters. This relationship may be thereason for IPT differences observed in assessed turtles, suggesting thatthe cellulolytic gut microflora is capable of degrading plant cell wallsand proteinwith acceptable digestibility efficiency under amixed diet.Thus, juveniles recruited to Gorgona's habitats seem to be consuminga better quality and more degradable mixed forage that increasesgrowth rates for more rapid achievement of sexual maturity. In regardto our experimental questions posed earlier, we can say the following:1) turtles in Gorgona consuming an omnivorous diet exhibited highfibre digestibilities (82 – 88%), similar to the 72 – 91% reported forgreen turtles in the Caribbean (Bjorndal, 1980); 2) NDF of 77% for themixed (omnivorous) diet suggests that intake does not necessarilyoccur rapidly. However, the animal might be compensated withnutrients for growth supplied by the high protein component of thediet and efficiency of the fibre content digestibility; 3) An IPT between14 and 32 days seems to be a reasonable time for an ectothermicanimal to efficiently digest meals composed of different types of food.Further research combining microbiological techniques, moleculartags, stable isotopes and satellite tracking, will contribute to furtherunderstanding the foraging ecology and importance of GorgonaNational Park as a transitional habitat for growth, performance andproductivity of East Pacific green turtles.

Acknowledgements

We thank the US National Fish and Wildlife Foundation (NFWF),The Rufford Small Grants Foundation (UK), the Colombian National

Parks Administrative Unit (UAESPNN), Centre for Research andEnvironmental Development (CIMAD, Colombia), the InternationalCentre for Tropical Agriculture (CIAT), the Zoo of Cali (Colombia), theR.E. Train Education for Nature (EFN), the University of West Indies(Barbados), and theWider Caribbean Sea Turtle Network (WIDECAST),for their financial, logistic and human contributions to this project.Our special gratitude to A. Pavia, M.J. Restrepo, J. Hoyos, A. Ruiz, J.Puertas, J. Rodriguez, L. Merizalde and other volunteers for theirenergy and enthusiasm proved while catching and processing turtlesduring the fieldwork. We also thank Dr. Fernando Gast from theColombian Biodiversity Research Institute Alexander von Humboldtand Dr. Fernando Castro from the Department of Zoology of the ValleUniversity for their support during this investigation. This study wasconducted under research permit DTSO 0029 from the UAESPNN(Colombia) and ethics approval BSCI /2003/04 from Monash Uni-versity (Australia). [SS]

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jembe.2008.04.009.

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24

CHAPTER 4. STOCK COMPOSITION OF THE

GREEN SEA TURTLE (CHELONIA MYDAS) IN

EASTERN PACIFIC FORAGING GROUNDS OF

GORGONA NATIONAL PARK IN COLOMBIA.



following:





Richard D. Reina



Date 12/2/09





authors.




25










Campus

Signature 1

Date 12/2/09

26

A manuscript submitted to Endangered Species Research

Stock composition of the green sea turtle (Chelonia mydas) in eastern Pacific foraging

grounds of Gorgona National Park in Colombia.

Diego F. Amorocho and Richard D. Reina*

School of Biological Sciences. Monash University, Clayton, Victoria 3800, Australia

* Corresponding author E mail: [email protected]

Running head: Stock composition of east Pacific green turtles

Key words: green sea turtle, foraging, developmental, juveniles, mitochondrial DNA, haplotype,

genetic diversity.

ABSTRACT

Mitochondrial DNA analyses have been useful for resolving maternal lineages and migratory

behaviour to foraging grounds of the loggerhead, hawksbill and green sea turtles. (Bowen et al.

1992). However, little is known about source rookeries and haplotype composition of foraging

green turtle aggregations in the south-eastern Pacific. We used mitochondrial DNA control

region sequences to identify the haplotype composition of 55 juvenile sexually unidentified green

sea turtles (Chelonia mydas) of unknown sex assessed during 45 days between October 2003 and

March 2004 in foraging grounds of Gorgona National Park in the Colombian Pacific. Amplified

fragments of the control region (457 bp) revealed seven haplotypes. The most common haplotype

was CMP4 observed in 84% of individuals, followed by CMP22 (5% of individuals). The

haplotype (h) and nucleotide (π) diversities were h = 0.300 ± 0.080 and π = 0.009 ± 0.005

27

respectively. Analyses of phylogenetic inference and genetic distance using maximum parsimony

methods showed the haplotypes clustered in a north-eastern Pacific geographic region and a

central-western Pacific geographic region. The genetic composition of this juvenile stock during

the sampling season revealed the presence of haplotypes from different distant breeding grounds

converging in foraging grounds at Gorgona. The information obtained here highlights the

importance of this protected area for conservation management of green turtles through marine

protected corridors connecting regional Marine Protected Areas of Ecuador, Colombia, Panama

and Costa Rica in the eastern Pacific Ocean.

INTRODUCTION Understanding the complex life cycle of endangered sea turtles has been a challenging research

priority to elucidate dispersal and migratory movements for the study of population biology and

for their full protection worldwide (Avise 1995; Bass et al. 2006; Bowen 1999; Bowen & Avise

1995; FitzSimmons et al. 1997a). This is quite difficult when animals migrating from different

nesting areas mix at distant foraging grounds. Direct sequencing of mitochondrial DNA

(mtDNA) control region provides great resolution for addressing questions about population

structure and identity of turtles in foraging grounds (Bowen 1995; Bowen et al. 1995; Bowen et

al. 1993; Dutton et al. 2008). The information can then be used to determine the geographic and

genetic origins of animals in a mixed foraging ground population.

The green sea turtle (Chelonia mydas) has a circumglobal distribution, occurring throughout

tropical and, to a lesser extent, subtropical waters of the Atlantic Ocean, Indian Ocean,

Mediterranean Sea and Pacific Ocean (Bjorndal 1985; Carr et al. 1978; Mortimer 1982). Green

turtles undertake complex movements and migrations through geographically disparate habitats,

28

and while they nest in more than 80 countries worldwide (Hirth 1997), it is thought that they

inhabit coastal waters of over 140 countries (NMFS 1998; Seminoff et al. 2002). Specific or

subspecific status has been given to green turtles, also known as black turtles (Chelonia mydas

agassizii) in the eastern Pacific Ocean; ranging from Baja California south to Peru and west to the

Revillagigedos Islands and Galapagos Archipelago (Green 1983; Pritchard 1971; Seminoff 2000).

Some authors have considered the black turtle either as a separate species, Chelonia agassizii,

distinguished by its greenish dark, vaulted shape and narrowed carapace (e.g. Cornelius 1982;

Pritchard 1999; Pritchard & Mortimer 1999), or a subspecies (Kamezaki & Matsui 1995).

Molecular phylogenetic studies tend to suggest that the black turtle Chelonia mydas agassizii is a

melanistic population within the Pacific clade of the genus Chelonia (Chassin-Noria et al. 2004;

Karl & Bowen 1999). The systematic status of the black turtle remains uncertain until today and

for the purpose of this study we considered the Pacific black turtle within the global Chelonia

mydas designation.

Long term mark-recapture studies of population dynamics in green turtle nesting areas show that

breeding turtles have a strong fidelity for their natal beach, to which they return to lay eggs

(Bowen 1995; Carr 1967; FitzSimmons et al. 1997b; Lahanas et al. 1994). This characteristic

“natal homing” behaviour has been confirmed with the use of mitochondrial DNA (mtDNA),

indicating that most nesting rookeries, also known as Management Units (MU) are distinct

populations. Spatially distinct MU typically contain unique maternal lineages, as would be

expected under the “natal homing” assumption (Bowen & Avise 1995). Based on molecular

sequence data it is possible to develop phylogenies that show broad areas of evolutionary

congruence between sea turtle species (Bowen & Karl 1997). Analysis of mtDNA genealogies in

the loggerhead turtle (Caretta caretta), hawksbill turtle (Eretmochelys imbricata), and green turtle

29

(Chelonia mydas) have demonstrated that most nesting aggregations are genetically distinct (Bass

1999; Bolten et al. 1998; Bowen & Avise 1995).

Foraging grounds aggregate animals from a variety of MUs, and analyses of mtDNA sequences

have been useful in identifying the source rookeries of turtles found in these feeding areas.

Feeding stocks have been characterized for loggerhead, hawksbill and green turtles. About 57%

of loggerhead turtles in the western Mediterranean feeding grounds are derived from nesting

populations in the south-eastern United States and about 43% are derived from Mediterranean

nesting populations (Bowen 1995). Large aggregations of loggerhead juveniles originally from

nesting beaches located in Japan comprised 95% (Bowen et al. 1995) of individuals recruited into

the Peninsula of Baja California feeding grounds 10.000 km away (Uchida & Teruya 1991) and

the remaining 5% came from Australian rookeries. Feeding hawksbill sea turtle populations in

Cuba and Puerto Rico are recruited from individuals of several distant Caribbean nesting areas

migrating over hundreds of kilometres (Bass 1999; Bowen et al. 1996). Green turtle feeding

stocks have been characterized in the Caribbean, the Brazilian Coast and the Hawaiian Islands.

Most of the green sea turtle juveniles feeding in the Bahamas are derived from Tortuguero, Costa

Rica so that the largest Caribbean nesting colony and other western Atlantic colonies are

represented approximately proportionally (Bjorndal et al. 2005; Lahanas et al. 1994). Most green

sea turtles feeding in the Brazilian coast come from South Atlantic and South African rookeries

(Naro-Maciel et al. 2007). Interestingly turtles at the Hawaiian feeding grounds come almost

exclusively from the French Frigate Shoals (Dutton et al. 2008). The composition of green turtle

feeding stocks in the eastern Pacific has not yet been well studied.

Molecular sequence data, tagging and satellite tracking studies have demonstrated that adult

female green turtle migrate great distances between specific nesting beaches and feeding grounds

30

at intervals of two to ten or more years (Bowen 1999; Luschi et al. 2003; Meylan 1982), usually

with high fidelity for feeding grounds throughout adulthood (Limpus et al. 1992). These feeding

grounds are usually shared with females from different rookeries of the same Ocean basin

(Pritchard 1976). Despite the importance of better understanding the genetic composition of

foraging grounds linked to breeding rookeries, little is known about the genetic diversity of green

sea turtle aggregations in feeding and developmental grounds of the eastern Pacific. Besides the

oceanic Archipelago of Galapagos in the south-eastern Pacific, immature green sea turtles are

found to the north, at the near shore Colombian island of Gorgona National Park. A well-

established stock largely composed of juveniles is present throughout the year in coral reefs and

surrounding waters of this Marine Protected Area (MPA). In contrast to the traditional

ontogenetic shift from a carnivorous to a herbivorous diet when turtles migrate from pelagic to

near shore waters for growth and sexual maturity, juvenile green turtles at Gorgona show an

opportunistic omnivorous feeding strategy to maximize energy acquisition in this neritic habitat (

Amorocho & Reina 2007; Amorocho & Reina 2008).

The importance of Gorgona as a common refuelling station in the eastern Pacific is highlighted by

the unusual role it plays as a transitional developmental ground for turtles. The foraging strategy

of animals, the population of almost exclusively juveniles, lack of recaptures of animals from year

to year and the absence of a resident or nesting population indicate that animals pass through

Gorgona on their way from one location to another. The striking morphological differences in

carapace shape and coloration lead us to think that these turtles are converging on this small

island from a variety of different regions (Figure 1). Thus, we asked where the juveniles of

different shapes and colours were recruited from and whether turtles at Gorgona are related to the

northern and southern nesting colonies in the eastern Pacific. In order to answer these questions

31

and to identify the geographic regions from which they might have come, an investigation of the

genetic composition of the transient population was necessary. Genetic information about the

green turtle populations can be used for more effective conservation management strategies in the

eastern Pacific region. Knowledge of the degree of demographic and evolutionary independence

among nesting rookeries is also important in order to track the movements of green turtles in

distant feeding grounds. The mtDNA control region, being maternally inherited, is characterized

by a rapid rate of evolution and higher proportion of genetic variance among populations. For

this reason the mtDNA control region is commonly used to define MUs (Bowen & Avise 1995;

Bowen & Karl 1997; Dethmers et al. 2006) and determine patterns of sea turtle dispersal (Dutton

et al. 2008; Naro-Maciel et al. 2007).

In this study we employed mtDNA control region sequences, to assess green sea turtles foraging

in Gorgona and to compare nucleotide composition and genetic variation with haplotypes

identified in other regions of the Pacific. We considered this information in the context of the

dispersal or migratory movements of individuals, contributing data useful for a greater

understanding of the green sea turtle ecology and the demographic structure based on

contemporary knowledge of their genome. The genetic identification of individuals composing a

mixed assemblage will allow governments and environmental NGOs to better enforce

conservation programs in feeding grounds and to track illegal trade of turtles markets along the

Pacific. If the genetic composition of turtle populations nesting, growing and foraging in tropical

islands of the central and south-eastern Pacific such as Gorgona (Colombia), Galapagos

(Ecuador), Coiba (Panama) and Coco (Costa Rica) is revealed, MPA can be linked to maintain

sea turtle connectivity and gene flow through managed marine corridors.

32

MATERIALS AND METHODS

Study site and sample collection Turtles were captured at night by hand using snorkel in water up to 7 m depth in coral reefs of

Gorgona National Park. (2º 56' - 3º 02' N, 78º10' - 78º13' W). This 9 km long and 2.5 km wide

island with a total protected area of 617 km² (including surrounding waters) is located 56 km

offshore the nearest mainland town of Guapi in the southern Colombian Pacific coast (Figure 2).

The island is surrounded by near-shore coral reefs where Pacific green turtles (Chelonia mydas)

are found throughout the year. Between October 2003 and March 2004, tissue samples were

collected from the shoulder of 55 animals, of which minimum curved carapace length (CCL) was

measured as described by Bolten (1999). Assessed turtles were weighed and double tagged in the

front flippers with Inconel 1005-681S tags (National Band & Tag Co.), following standard

techniques (Balazs 1999). Tissue samples were stored in a lysis buffer (0.1M Tris, 0.1M EDTA,

0.01M NaCl and 0.5% SDS, Ambrose Monell cryo collection American Museum of Natural

History) and processed in the Laboratory of Molecular Biology and Tissue Bank of the

Colombian Alexander von Humboldt Biodiversity Research Institute (IAvH) in Cali, Colombia.

DNA extraction, PCR amplification and sequencing

DNA was extracted from tissue samples using a Qiagen DNeasy kit (Qiagen, Germantown MD,

USA) and DNA concentration and quality was visualised by electrophoresis of 5 μl on a 0.8%

agarose gel stained with 500 μg/μl EtBr. We targeted a 550 base pair fragment of the mtDNA

control region using primers LTCM2 and HDCM2 (Lahanas et al. 1994). For the polymerase

chain reaction (PCR) we used 2 μl of template DNA in 25 μl reaction volumes containing 50 ng

of genomic DNA 1 μl of Taq polymerase (Perkin-Elmer/Cetus, Norwalk, PA) 10 μM of each

33

primer, 5 μM dNTPs (Deoxynucleotide Triphosphates) 25 μM MgCl2 and Buffer 10X (KCl

500mM and 200mM Tris-HCl, pH 8.4). The denaturing, annealing and extension steps consisted

of 35 cycles using a thermal controller (PTC-100, MJ Research, Waltham MA, USA) applying 20

s at 94 °C 20 s at 53 °C and 20 s at 72 °C. The initial denaturing (94 °C) and last extension (72

°C) cycles were of 2 minutes each (Saiki et al. 1998). A sample of 2 μl of the obtained PCR

products were run on 1% agarose electrophoresis gel at ~ 60 °C until the bromophenol blue dye

marker reached the edge of the plate. We used the Poly Ethylene Glycol (PEG) method (20%

PEG 8000 2.5 M NaCl) to clean up the PCR products prior to sequencing both forward and

reverse in a Multi - Color Capillary Electrophoresis sequencer (ABI 310 - 3100) at Macrogen Inc

(Seoul, Korea).

Mitochondrial DNA haplotype characterization and data analysis

The mtDNA sequences were edited and assembled using Chromas Pro v1.34 (Technelysium Pty

Ltd, Tewantin QLD, Australia) and trimmed before alignment with CLUSTAL X v1.74

(Thompson et al. 1997). Each nucleotide change encountered in the individuals’ sequences was

considered a different haplotype. Haplotypes encountered in Gorgona National Park were

assigned a temporary code (GPC) and compared with other Pacific Ocean sea turtle haplotypes

reported in the NCBI-GenBank (www.ncbi.nlm.nih.gov) and the Marine Turtle Research Program

database (http://swfsc.noaa.gov/textblock.aspx?Division=PRD&ParentMenuId=212&id=11212).

To assess the genetic diversity of the overall turtle population we estimated haplotype (h) and

nucleotide (π) diversities (Nei 1987) using ARLEQUIN v3.1 (Excoffier et al. 2006). Analysis of

molecular variance (AMOVA) to determine the variation within and among groups in both

western and eastern Pacific Ocean was obtained with using Tamura – Nei model based on

34

nucleotide substitution. Indirect measurements of genetic distances (Fst), were used to estimate

diversity within and among ocean groups based on the frequencies of identified haplotypes (Fst: 1

/ 1 + 4 Nm). Gene flow was estimated based on the Fst (Nm: 1/4 (1/Fst) - 1) method proposed by

Wright (1951). Phylogenetic analyses were conducted using the seven haplotypes found in

Gorgona and a set of sequences selected for Pacific green sea turtles, including haplotypes from

eastern and western Pacific. Sequences of the species Natator depressus and Caretta caretta

were used as outgroups. Maximum parsimony analyses were conducted using the Mega v3.1

program (Kumar et al. 2004). Searches were conducted with CNI (close-neighbour-interchange)

with 100 random addition replicates with 500 bootstrapping replicates.

RESULTS The mean CCL of 55 sampled turtles was 61. 2 ± S.D. 8.2 cm (ranging from 42.7 to 77.6 cm),

with 47 juveniles (< 70 cm CCL), 7 large juveniles (70 - 75 cm CCL) and one adult (> 75 cm

CCL).

Haplotypes identification

A total of seven haplotypes were detected from the mtDNA control region (D-Loop) of the 55

green turtles sampled in Gorgona’s feeding grounds (Table 1). From the 457 bp sequence of the

mtDNA control region there were 431 (94%) invariant sites and 26 (6%) polymorphic sites. The

Gorgona haplotypes were compared with sequences from the Marine Turtle Research Program

data base 100% identity was used to designate the Gorgona haplotypes to known types in the

database. The most common haplotype was CMP4, found in 83% of the sampled population, and

together with CMP8 (2%) correspond to the characteristic black turtles (known locally as tortuga

35

prieta) with greenish dark colour and vaulted carapace that breed in Michoacán state and

Revillagigedos Archipelago (Mexico). Additionally we identified haplotypes CMP15 (2%) and

CMP17 (2%) in Gorgona. These latter two haplotypes (together with CMP4 and CMP8) have

also been identified in the Galapagos rookeries (P. Zarate pers. comm., P. H. Dutton et al. unpubl

data). Haplotypes CMP21 (2%) and CMP22 (5%) have been reported for the Micronesia and Fiji

cluster (Dutton et al. unpubl data) and the Australasian region (Dethmers et al. 2006). The

haplotype referred to as GPC7 (4%) had not been reported in the NCBI-GenBank database but

seems to be the same sequence as CMP97 present in Galapagos and the Indo Pacific (P. Dutton,

A. Abreu pers comm.) Geographical comparisons between haplotypes previously described in

nesting and foraging grounds in the Pacific and in Gorgona developmental habitats are shown in

Table 2.

mtDNA genetic diversity and phylogenetic stock composition

Haplotype diversity (h) was 0.300 +/- 0.080 and nucleotide diversity (π) was 0.009 +/- 0.005 for

the overall 55 sampled turtles (Table 3). The nucleotide composition of the sequences was biased

towards A-T bases: C = 20.23%, T = 31.41%, A = 34.51% and G = 13.85%.

The most parsimonious tree of the seven haplotypes of Gorgona, using loggerhead turtle (Caretta

caretta) and flatback turtle (Natator depressus) as outgroups, revealed two distinct clades (Figure

3a). Clade 1 contains four haplotypes with great similitude among them and with other

haplotypes reported for the eastern and central Pacific: CMP4 (GPC1) and CMP8 (GPC3) have

been reported in Mexico (Michoacán and Revillagigedos Island) and Ecuador (Galapagos

Islands); CMP15 (GPC2) and CMP17 (GPC4) are described for the central and eastern Pacific

without precise natal origin assignation yet. Clade 2 contains three very similar haplotypes that

are identical to other haplotypes identified in the western Pacific: CMP22 (GPC5) and CMP21

36

(GPC6). Haplotype GPC7 (Genbank Accession Number = FJ268479) is different from all the

others but has some similarity with the GPC6 and GPC5 haplotypes present in the Australasian

region. The same haplotype and geographical distribution pattern is observed in the global most

parsimonious tree on which sequences of Chelonia mydas from other sites in the Pacific and

Atlantic were incorporated maintaining the same outgroups. The resulted tree separated four

clades. Clade 1: comprises eastern Pacific haplotypes; Clade 2 and Clade 3 include the

Australasian region; Clade 2 separates the two groups of seven haplotypes from Gorgona. Clade

4 corresponds to Chelonia mydas from the Atlantic basin which haplotypes are highly

differentiated from those in the Pacific. The genetic distance among groups (within the same

clades) was small, but between the two separated groups (clades) it was much higher. Haplotypes

CMP4, CMP8, CMP15, CMP17 (Clade 1) and CMP21, CMP22, GPC7 (Clade 3) are located on

opposite sides of the cladogram (Figure 4). There are characteristic melanistic differences

observed in carapaces of individuals recruited to feeding grounds of Gorgona from Michoacán in

the north-eastern Pacific (CMP4, CMP8), Fiji-Micronesia, central (CMP15, CMP17) and western

(CMP21, CMP22, CMP97). Carapace colours for CMP4, CMP8 were green- black while the

CMP15, CMP17 included dark yellow, light brown and dark orange scutes; and CMP21, CMP22,

CMP97 exhibited variations from pale green to dark yellow or caramel (Figure 1).

The AMOVA analysis indicate that 97% of genetic variation (Fst: 0.970; P= 0.000) accounted for

differences among western and eastern Pacific haplotypes and 2.95% for differences within each

of the two groups when considered separately. In the eastern group h (0.119 ± 0.063) and π

(0.000 ± 0.000) were low, and this might be a consequence of the reduced number of haplotypes

(four) present in 49 turtles of which 46 have the same haplotype (CMP4). In contrast, the western

37

Pacific exhibited a relative high value of h (0.733 ± 0.155) and π (0.009 ± 0.006); maybe because

there was more variation in the small group of six turtles where three haplotypes (two animals per

haplotype) were identified (Table 4). The low value of Nm (0.006), suggests a lack of gene flow

between western and eastern groups of haplotypes detected in Gorgona. This result is consistent

with the high estimated value of Fst (0.970) when both groups are compared. Strong genetic

differentiation is observed at Gorgona’s feeding stock composed of individuals with mixed

haplotypes from eastern and western Pacific regions.

DISCUSSION Results indicate that green turtle stock at feeding grounds of Gorgona National Park is composed

of juveniles recruited from different sites in the northern, central and western Pacific. This new

information sheds light on the species genetic structure and population arrangements in the

Pacific basin contributing important knowledge for conservation strategies.

Genetic composition and diversity of Gorgona’s stock

The observed bias towards A-T bases has been reported as common in Chelonia. mydas and other

sea turtles (Chassim et al. 1998; Karam 1997). The seven haplotypes identified in Gorgona are

clearly separated in two different geographic groups from both sides of the Pacific, demonstrating

the importance of this near-shore island to maintain genetic diversity of green turtle populations in

the Ocean basin. The presence in Gorgona of haplotypes identified in the Micronesia- Fiji region,

Galapagos and Mexico, indicates that these turtles are migrating from or to very distant places,

which is critically important for the species conservation management throughout their migratory

routes.

38

The genetic diversity estimated for the Gorgona foraging grounds is within the ranges for green

turtle nesting rookeries and feeding aggregations in other Pacific (Chassim et al. 1998; Dethmers

et al. 2006; Dutton et al. 2008; FitzSimmons et al. 1997b) and Atlantic sites (Bass et al. 2006;

Bjorndal et al. 2005; Bowen et al. 1992; Encalada et al. 1996; Lahanas et al. 1994). The overall

haplotype diversity (h = 0.300) and nucleotide (π = 080) diversities were relatively low, due to the

conspicuous dominance of Michoacan’s haplotype CMP4, present in 46 (83%) of the 55 sampled

turtles. Gorgona hosts turtles of distant and genetically diverse origins, while those in Michoacán,

Mexico, are more homogeneous and closely related among themselves (CMP4, CMP8). Despite

the limited number of sampled individuals (55) and comparatively low number of haplotypes

(seven), high nucleotide differences were observed within the Gorgona aggregation (Table 3).

The π value was also high compared to other localities in the Atlantic such as Brazil (Ubatuba

0.002; Almofala 0.006), Tortuguero in Costa Rica (0.003) and other sites in the wider Caribbean

(0.000 - 0.005) (see Table 3). The number of haplotypes and magnitude of changes in nucleotide

sequences indicate that Gorgona is an important place for green turtles in order to maintain

population diversity and genetic variability in the western and eastern Pacific. The AMOVA

results suggest that genetic variation is distributed in a heterogeneous way among eastern and

western Pacific, indicating high levels of genetic differentiation in turtles recruited to Gorgona’s

feeding grounds.

Dispersal, recruitment and migratory behaviour of Gorgona stock

Site fidelity of juveniles at Gorgona is not strong, only three recaptures have been reported from

more than 400 individuals tagged in the last four years ( Amorocho & Rodriguez-Zuluaga 2008).

Our findings reinforce what has been suggested in other sea turtle studies - that the recruitment

39

into feeding grounds is influenced by oceanic mixing of individuals during the 2 – 3 year pelagic

stage (Bass et al. 2006; Velez-Zuazo et al. 2008). Equatorial currents may be an important

vehicle for dispersal of green turtle post-hatchlings from western to eastern Pacific, and vice

versa. Once arriving to neritic areas such as Gorgona, juveniles spend some time recovering from

the transoceanic journey and storing resources to continue travelling to further developmental and

mating grounds. However, the abundance of the black turtle or so called tortuga prieta (CPMP 4

haplotype) indicates the existence of marine corridors, or paths connecting Michoacán State

(Mexico) rookeries, all of which comprise the same MU (Chassin-Noria et al. 2004), with the

foraging grounds of Gorgona and Galapagos (Seminoff et al. 2008) in south-eastern Pacific.

Gene and nucleotide diversity estimated at Gorgona provides new information to describe the

genetic structure of a diverse green turtle juvenile stock and how it is influenced by the

demographic composition of foraging grounds along MPA in the eastern Pacific.

Some evidence on melanistic similarities and size class between Gorgona and Galapagos turtles

might suggest that Galapagos could be the next destination after large juveniles from western and

eastern Pacific abandons Colombian foraging grounds. This might be possible considering that

there are migratory corridors for Galapagos post-nesting green turtles including oceanic migration

to Central America, residency within the Galapagos and movement into oceanic waters south west

of the Galapagos (Seminoff et al. 2008). Satellite or GPS telemetry will enormously contribute to

better understanding the dispersal and migratory movements of green turtle juveniles after they

leave Gorgona. The mapped routes of satellite tracked animals supported by flipper tagging data

would be a useful tool in designing accurate plans for the species conservation management in the

Pacific. In order to fully identify the geographically nesting origin of reported haplotypes in

40

Gorgona, molecular assignments need to be conducted to determine the percentage and

contribution of haplotypes from each Pacific site to Gorgona’s green turtle aggregation. This

information would be important for Colombian environmental authorities to better protect

Gorgona marine habitats and to advance in the establishment of multinational strategies to ensure

the survival of sea turtles in the Pacific Ocean.

Conservation implications

The conservation of the Gorgona’s juvenile stock is essential to maintain diversity among green

sea turtles in the Pacific basin. The convergence of turtles from distant nesting rookeries in this

MPA and the role it plays as a refuelling station to provide shelter and food to immature green

turtles from the eastern and western Pacific is important for regional management.

The high genetic differentiation (Fst = 0.975) within Gorgona foraging stock suggests an apparent

disadvantage in terms of conservation. For management purposes it is much better to have a

stock with low values of Fst, which means that in a more homogenous population, the extinction

of one colony would not necessary put at risk the entire population or affect the genetic structure

of the entire MU. This could be considered under critical circumstances for conservation

management decisions. If genetic variability is high as in Gorgona’s stock, to determine which

foraging ground of the eastern Pacific contains the greater genetic diversity is a priority. From the

genetic perspective, this is a useful criterion for identification of main concern sites where green

sea turtle conservation plans along the eastern Pacific region need to be strengthen or initiated.

These findings are valuable inputs for development of sea turtle research-driven management

strategies in the eastern tropical Pacific corridor (Corredor Marino del Pacífico Oriental

41

Tropical). This is a transnational alliance carried out by Central and South American

governments through a conservation network of MPA. The conservation goal is to investigate

and promote the biological connectivity among the islands of Galapagos (Ecuador), Malpelo and

Gorgona (Colombia), Coiba (Panama), and Coco (Costa Rica), for regional management of

threatened migratory species with special attention to whales, sharks and marine turtles. The

results of this study will be used to the design and implementation of the Regional Plan currently

developed by the network of sea turtle experts from countries integrating the eastern tropical

Pacific corridor.

42

ACKNOWLEDGEMENTS We thank Monash University, US National Fish and Wildlife Foundation (NFWF), Rufford

Foundation, Chelonian Foundation, Colombian National Parks Administrative Unit (UAESPNN),

Centre for Research and Environmental Development (CIMAD), University of West Indies,

Wider Caribbean Sea Turtle Network (WIDECAST), and many volunteers for their financial,

logistic and human contributions to this project. Our gratitude goes to F. Gast, J.D. Palacio, C.

Villafañe and L. Camacho from the Molecular Biology and Tissue Bank of the National

Biodiversity Research Institute Alexander von Humboldt for sharing the facilities and equipment

for DNA analysis, and advice during the lab work. Many thanks to A. Abreu, P. Sunnucks and A.

Jaramillo, for their comments and contributions to the manuscript. Finally, many thanks to F.

Arias and the Colombian Marine Research Institute (INVEMAR), which acted as national

scientific authority, backing the investigation under the Andean Decision 391 and Colombian law

for access to genetic resources. This study was conducted under research permit DTSO 0029

from the UAESPNN, contract for access to genetic resources issued by the Colombian Ministry of

the Environment 28/03/2007 and ethics approval BSCI/2003/04 from Monash University.

43

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FIGURES AND TABLES

Figure 1. Chelonia mydas. Differences in colour and carapace shape of green sea turtle

juveniles corresponding to western CMP21 (GPC6) and eastern Pacific CMP4 (GPC1)

haplotypes recruited at Gorgona National Park in Colombia. Photo: Javier Rodriguez-

Zuluaga.

Figure 2. Gorgona National Park in the Colombian Pacific. Turtles were caught by hand

in the coral reefs of La Azufrada and Playa Blanca study sites.

Figure 3. Chelonia mydas. Maximum Parsimony phylogenetic tree of Gorgona’s green

turtle stock haplotypes. Cladogram is separated into two distinct groups composed of

individuals with origin in the eastern (CMP4, CMP8, CMP15, CMP17) and western

(CMP21, CMP22, GPC7) Pacific. Caretta caretta and Natator depressus sea turtles were

used as out-groups. Numbers at branches are bootstrap values.

Figure 4. Chelonia mydas. Most parsimonious tree for sequences of haplotypes found in

Gorgona GPC (underlined) with their GeneBank assignation (CMP, E). All haplotypes

were distributed in four separated clades (1 - 4) distributed across the Pacific and Atlantic

Oceans. Caretta caretta and Natator depressus sea turtles were used as out-groups.

51

Figure 1.

52

Figure 2.

53

Figure 3.

54

Figure 4.

55

Table 1. Chelonia mydas. mtDNA control region haplotype frequencies of 55 green

turtles at Gorgona National Park (GNP) in the Colombian Pacific. A provisional code

GPC (Green Pacific Colombia) has been used to describe Gorgona haplotypes. CMP

corresponds to Chelonia Mydas Pacific listed in NOAA and NCBI gene banks.

Haplotypes Frequency # of individuals CMP4 (GPC1) 0.836 46 CMP15 (GPC2) 0.018 1 CMP8 (GPC3) 0.018 1 CMP17 (GPC4) 0.018 1 CMP22 GPC5) 0.054 3 CMP21 (GPC6) 0.018 1 CMP97 (GPC7) 0.036 2 Total 55

56

Table 2. Chelonia mydas. Composition of green turtle mtDNA haplotypes compared

among nesting, feeding and developmental population data in the Pacific. Adapted from

Dutton et al. 2008 (Chassin-Noria et al. 2004; Dethmers et al. 2006; Dutton et al. unpubl.

data).

Nesting Grounds Haplotype- Eastern Pacific Western Pacific

Eastern Pacific Feeding Grounds

Central North Pacific

Total

Mexico Michoacan

Mexico Revillagigedos

Galapagos Australasia Ellato Atoll

Australasia Ngulu Atoll

Colombia Gorgona

Hawaii strandings

CMP4 (GPC1)

82 23 95 46 1 247

CMP15 (GPC2)

3 1 4

CMP8 (GPC3)

2 1 3

CMP17 (GPC4)

1 1

CMP2 (GPC5)

3 3

CMP21 (GPC6)

2 10 1 13

CMP97 (GPC7)

2 2

Total 84 26 95 2 10 55 1 273

57

Table 3. Chelonia mydas. Comparison of mtDNA control region sequence diversities in

Gorgona green sea turtle (feeding grounds and other worldwide populations, measured as

haplotype diversity (h) and nucleotide diversity (π) ± standard error. Sample size (n)

refers to number of animals sampled.

Geographic site No of

Haplotypes h π n Reference

Gorgona / Colombia

7 0.30 ± 0.080 0.009 ± 0.005 55 This study

Michoacán / Mexico

5 0.48 ± 0.040 0.003 ± 0.002 123 (Chassin-Noria et al., 2004)

Australasia region

25 0.88 ± 0.010 0.041 ± 0.020 714 (Dethmers et al., 2006)

Almofala / Brazil

13 0.71 ± 0.030 0.006 ± 0.000 117 (Naro-Maciel et al., 2007)

Ubatuba / Brazil 10 0.44 ± 0.556 0.002 ± 0.001 113 (Naro-Maciel et al., 2007)

Wider Caribbean Florida Costa Rica Isla Aves Surinam

11 10 8

15

0.61 ± 0.103 0.20 ± 0.154 0.25 ± 0.194 0.25 ± 0.141

0.001 ± 0.001 0.000 ± 0.000 0.005 ± 0.001 0.000 ± 0.000

44

(Lahanas et al., 1994) (Encalada et al., 1996)

Tortuguero / Costa Rica

5 0.16 ± 0.020 0.003 ± 0.002 433 (Bjorndal et al., 2005)

58

Table 4. Chelonia mydas. Molecular variance (%) of 55 green sea turtles compared

among and between eastern and western Pacific groups of haplotypes identified at

Gorgona National Park.

Source of variation df Sum of squares Variance components Percentage of variation Among groups 1 98.904 922.499 97.050 within groups 53 14.878 0.2807 2.954 total 54 113.782 950.570

Fixation index Fst: 0.975

59

CHAPTER 5. GENERAL DISCUSSION AND

CONCLUSIONS

Green turtles are among the larger herbivorous vertebrates grazing on seagrasses

and/or algae. This feeding strategy is commonly observed in the species along the

Caribbean, Australia, northern and central America (Bjorndal, 1980; Limpus, et al.,

1994; Mortimer, 1982; Seminoff, 2000), but is less common in some sites of the south

eastern Pacific (Green, Ortiz, 1982; Hays Brown, Brown, 1982). In this study I

provided new data about the feeding ecology and genetic composition of immature

green turtle stock occurring in the Colombian Pacific. This information is key for the

implementation of conservation management strategies to protect sea turtles along the

Eastern Tropical Pacific Corridor, a multinational effort to preserve endangered

species in the eastern Pacific Ocean.

5.1 Shifting feeding diets

As juvenile green turtles grow and mature, their movement from pelagic to neritic

waters is typically accompanied by a shift from a carnivorous to generally

herbivorous diet. However, my studies show that this does not occur in Gorgona’s

feeding grounds. A more opportunistic diet is consumed by animals converging in

this recruitment area and an explanation for this strategic shift in feeding behaviour

could be the absence of sea grass beds, as well as low diversity and abundance of

algae in the Colombian Pacific (Bula-Meyer, 1995). These two main food items

abundant for green turtles in other parts of the world are replaced by red mangrove

(Rhizophora mangle) in Gorgona, as has been observed in Australia (Limpus, Limpus,

60

2000). Alternatively, the diet of Gorgona’s turtles may be due to the oceanographic

conditions and nutrient richness provided by the Humboldt current in the south east

Pacific. The cold waters in south eastern America lead to abundant fish and other

marine animals like tunicates (Salps sp) frequently consumed by green turtles in

Gorgona’s feeding grounds. We must also acknowledge the benefits offered by a

high protein diet for juveniles to gain the necessary energy for continuing the journey

towards sexual maturity throughout developmental grounds in the Pacific basin.

5.2 Nutritional benefits of an omnivorous diet

The use of variable food sources (such as tunicates and terrestrial vegetative matter)

correspond to an omnivorous feeding behaviour which differs from the ontogenetic

trend of immature green turtles (Chelonia mydas) to become chiefly herbivorous

when entering near-shore feeding habitats. This involves a change in the foraging

habits very different from grazing on sea grasses or algae. Perhaps foraging patterns

in juveniles are driven more by the foraging stock geographical distribution, type of

habitat and food availability than by ontogenetic changes experienced by animals

when they enter neritic shallow waters. This relationship may be the reason for IPT

differences observed in assessed turtles, suggesting that the cellulolytic gut microflora

is capable of degrading plant cell walls and protein with acceptable digestibility

efficiency in a mixed diet. Thus, juveniles recruited to Gorgona’s habitats seem to be

consuming a better quality and more degradable mixed forage that increases growth

rates for more rapid achievement of sexual maturity. Turtles in Gorgona consuming

an omnivorous diet exhibited high fibre digestibilities (82 – 88 %), similar to the 72 –

91% reported for green turtles in the Caribbean (Bjorndal, 1980). NDF of 77% for the

mixed (omnivorous) diet suggests that intake does not necessarily occur rapidly.

61

However, the animal might be compensated with nutrients for growth supplied by the

high protein component of the diet and efficiency of the fibre content digestibility.

Thus, an IPT between 14 and 32 days seems to be a reasonable time for an

ectothermic animal to efficiently digest meals composed of different types of food.

5.3 Gorgona: a stopover and marine protected area

The significance of the omnivorous dietary composition in the context of food

availability and energy supply highlights the role of Gorgona Island as a refuelling

station for immature green turtles migrating along this part of the south eastern Pacific

coastline. Results from this study show how the transitional feeding grounds of

Gorgona National Park provide nutritional opportunities that benefit turtles’ digestive

efficiency that can be translated into growth and fitness. In contrast to the traditional

ontogenetic shift from a carnivorous to a herbivorous diet when turtles migrate from

pelagic to near shore waters for growth and sexual maturity, juvenile green turtles at

Gorgona show an opportunistically omnivorous feeding strategy to maximize energy

acquisition in this neritic habitat. The foraging strategy of animals, the population of

almost exclusively juveniles, lack of recaptures of animals from year to year and the

absence of a resident or nesting population indicate that animals pass through

Gorgona on their way from one location to another. The striking morphological

differences in carapace shape and coloration suggest that these turtles are converging

on this small island from a variety of different regions. This suggestion was supported

in this study through the genetic identification of 7 haplotypes present in 55 assessed

turtles. These haplotypes has been previously described in foraging and nesting

rookeries of the northern, central and western Pacific Ocean. Green turtles found at

Gorgona are more genetically diverse than at other feeding grounds, such as those

62

sampled around the Hawaiian Islands that are principally of one genetic stock derived

from the nesting population of French Frigate Shoals located in the middle of the

Hawaiian Archipelago (Dutton, et al., 2008). Our results indicate that green turtles in

Colombia do not include haplotypes from the north-central Pacific (Dutton, et al.,

2008), but do incorporate haplotypes reported by Dethmers et al., (2006) from the

Australasian populations (CMP20, CMP21 and CMP22) and Chassin-Noria et al.,

(2004) from the north-eastern Pacific coast of Mexico. This genetic composition

observed in Gorgona supports the suggestion by Dethmers et al., (2006) of a historical

divergence between green turtle populations in the Pacific Ocean and those in the

Indian Ocean and south-east Asian region..

To ensure that Gorgona remains available as a diverse and important feeding and

transitional habitat for green turtles, it is necessary to address the abundance of debris

and quality of food items ingested by omnivorous juveniles and to reduce by-catch in

the protected habitats. The main cause of current species depletion in the eastern

Pacific is the incidental capture of turtles in fishing gear, particularly long lines and

gillnets (Cheng, Chien, 1997; Hall, et al., 2000; Polovina, et al., 2003). Marine

protected areas such as Gorgona play a significant role in addressing these

conservation issues and are important for the preservation of endangered species.

Sound research, as well as systematic and long term monitoring need to continue and

be strengthened in Gorgona National Park for appropriate conservation of migratory

green sea turtles on this side of the eastern Pacific Ocean.

63

5.4 Recommendations for further research

Further research should focus on the relationship between food selection, nutritional

value of a mixed diet intake examining intake passage rate (IPT) and the effect of diet

quality (through nutrient limitation), on the productivity and permanence of foraging

sea turtles. For this purpose, combining microbiological techniques, molecular tags,

and stable isotopes will allow better understanding of the foraging ecology and

importance of Gorgona National Park as a transitional habitat for growth,

performance and productivity of green turtles in the eastern Pacific. Satellite or GPS

telemetry will enormously contribute to elucidate the dispersal and migratory

movements of green turtle juveniles after they leave Gorgona. The mapped routes of

satellite tracked animals supported by flipper tagging data would be a useful tool in

designing accurate plans for the species conservation management in the Pacific. In

order to fully identify the geographically nesting origin of reported haplotypes in

Gorgona, molecular assignments need to be conducted. Mixed stock analysis will be

the next step to determine the percentage and contribution of haplotypes from each

site to Gorgona’s green turtle population. This information would be critical for

Colombian Environmental Authorities to better protect Gorgona marine habitats and

to advance in the establishment of multinational strategies to ensure the survival of

sea turtles in the Pacific Ocean.

64

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