Worldwide mtDNA phylogeography and diversity of pilot whale species (Globicephala spp)

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Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity

 Abstract

Spinner dolphins (Stenella longirostris) are found in apparently relatively small and discrete communities around many islands throughout the Pacific. However, the boundaries of these communities on the scale of a dolphin’s lifespan or across generations are unknown. Here a combined demographic and genetic approach is reported to describe the isolation and interchange of insular spinner dolphins among island communities of the Society Archipelago, French Polynesia. Dorsal fin photographs for individual identification and biopsy samples for genetic analyses (n = 154) were collected from six island communities during 189 small-boat surveys over three years. Capture-recapture analyses at Moorea (the primary study site), based on long-term observations of distinctively marked individuals and microsatellite genotypes (12 loci), indicated a local community of about 150 dolphins. This community appeared relatively closed on an intra-generational scale, as confirmed by re-sightings of individuals across 15 years. Surveys around neighbouring islands indicated the presence of similar distinct communities, likely to follow similar demographic patterns to Moorea, with relatively low level of interchange between communities. Overall, significant differentiation at both mitochondrial and nuclear levels indicates restricted gene flow among neighbouring communities, although some individual movement was documented. High levels of insular mtDNA genetic diversity (Nef ~ 100,000) contrasted with demographic characteristics. No evidence of a bottleneck was found in microsatellite allele frequencies or mtDNA haplotypes, discounting the possibility of a recent founder effect. Instead, this genetic pattern suggests that it is the result of metapopulation structure, based on numerous insular communities evolutionarily connected through male and female gene flow.

Introduction

Dolphins are often found in relatively small and apparently discrete coastal or insular communities that are assumed to exhibit genetic exchange with neighbouring communities or larger pelagic populations (e.g., Wells 2003). However, with the exception of a few populations that have been the focus of extensive studies, the social and reproductive boundaries of the communities and the extent of demographic and genetic interchange remain unknown.
Demographic approaches, based principally on photographic documentation of naturally marked individuals (i.e., photo-identification), can provide valuable information on social relationships and local abundance. These methods are limited, however, when assessing large-scale geographic structure and population dynamics that extend across generations. On the other hand, evolutionary approaches are often aimed primarily at estimating population genetic parameters but do not provide a clear distinction between the relative importance of contemporary and historical processes. Combining demographic and genetic methods can help overcome the limitations of each (Lande 1988).
Spinner dolphins (Stenella longirostris) pose an interesting challenge to the description of community structure. The species has a worldwide circum-tropical and subtropical distribution (Perrin & Gilpatrick 1994) within which four subspecies have been described based on morphological characters, distribution and habitat preferences (Perrin & Gilpatrick 1994, Perrin et al. 1999): the eastern spinner (Stenella longirostris orientalis), the Central American spinner (S. l. centroamericana), the dwarf spinner (S. l. roseiventris) and Gray’s spinner (S. l. longirostris) (Figure 2.1a). The distribution of the Central American spinner is limited to waters of the west coast of southern Mexico to the Gulf of Panama, while the dwarf spinner is only found in the Gulf of Thailand and Timor Sea (Perrin et al. 1999). In the Eastern Tropical Pacific (ETP), the eastern spinner and the whitebelly spinner (an apparent hybrid form between S. l. orientalis and S. l. longirostris) form large, pelagic, mixed-species aggregations with spotted dolphins (Stenella attenuata) and yellow fin tuna (Thunnus albacares). Due to this association with tuna, millions of these dolphins have been killed as by-catch in the yellow-fin tuna purse-seine fishery during the last four decades (Wade & Gerrodette 1993). Concerns about the impact of this large-scale dolphin mortality led to numerous studies on various aspects of their biology, including genetic diversity and population structure (e.g. Galver 2002), mating strategies (Perrin & Mesnick 2003), and abundance (e.g. Wade & Gerrodette 1993). Figure 2.1. The location and details of the study area in relationship to worldwide distribution of spinner dolphins. (a) Global distribution of spinner dolphin subspecies (from Galver 2002; A = Stenella longirostris longirostris, B = S.l. orientalis and whitebelly spinner dolphin, C = S.l. centroamericana, D = S.l. roseiventris. (b) Map of French Polynesia, including the Society Islands and Nuku Hiva in the Marquesas Islands. (c) Map of the Society Islands; arrows indicate movement of individuals between islands based on photo-identification (full line) and genotyping (dashed line). Number of events represented by each arrow is given (d) Map of Moorea, the primary study site.
In contrast to the pelagic distribution of the eastern and whitebelly spinner, Gray’s spinner dolphin is primarily insular in habitat preference (Perrin & Gilpatrick 1994). Although absent from the ETP, its geographic distribution is much greater than the distribution of the other sub-species, extending across the tropical and subtropical waters of the Atlantic, Indian and Pacific Oceans (Figure 2.1a). Much of what is known about the population dynamics of S. l. longirostris has been derived from a few island locations: behavioural observation and photo-identification at the Big Island, Oahu and Midway Atoll in Hawaii (Norris et al. 1994, Lammers 2004, Karczmarski et al. 2005), Fernando de Noronha in Brazil (Silva-Jr et al. 2005) and Moorea, in the Society Archipelago of French Polynesia (Poole 1995). These studies reveal that insular Gray’s spinner dolphins (hereafter referred to as spinner dolphins) follow a similar daily cycle at each location; during the day, they rest and socialise in inshore habitats and at dusk, they move offshore where they feed on squid, shrimp and mesopelagic fish. Demography and social organisation, on the other hand, appear to be substantially different in each of the studies locations.
Around the Big Island of Hawaii, where the dolphins use specific bays and shallow reefs during the daytime, Norris et al. (1994) found a ‘fission-fusion’ model of social organisation, with groups forming and separating from day to day. Because of the regular identification of new individuals in the resting groups, the authors concluded that the dolphins observed around this island form an open population of more than 1,000 individuals (Norris et al. 1994). More recently, Karczmarski et al. (2005) has described a very different social organisation of spinner dolphins at the remote atoll of Midway, in the far-western leeward Hawaiian Islands. This population of about 200 individuals was found to be closed with respect to immigration/emigration (or nearly so), with strong geographic fidelity and no obvious fission-fusion (Karczmarski et al. 2005).
In the Society Archipelago of French Polynesia, Poole (1995) described an intermediate form of social organisation. Around the island of Moorea, the primary study site (Figure 2.1), groups of spinners rest and socialise in a series of 10 pass/bay complexes (Figure 2.1d). These groups follow the same fission-fusion model of social organisation observed at the Big Island, with day to day fluidity in group composition (Poole 1995). However, similar to Midway Atoll, photoidentification surveys over six years indicated that Moorea’s spinner dolphins were year-round long-term residents forming a small and apparently closed community, although some low-level of interchange was documented with the sister island of Tahiti, just 17 km away (Poole 1995).
These island specific studies revealed important features of the behavioural ecology of insular spinner dolphins, but left unanswered several crucial questions related to genetic diversity and population dynamics: Do spinner dolphins typically form relatively closed island communities distinct from one another, as suggested by observations at Moorea and Midway? What are the social and genetic boundaries of insular spinner dolphin communities? Is there any interchange of dolphins between island communities and at what frequency? Are island communities formed by colonisation events followed by isolation or do they maintain connectivity to ‘parent’ populations, forming large metapopulations?
To address these questions, evolutionary and demographic approaches were combined, using microsatellite genotyping and mitochondrial DNA sequences obtained from biopsy samples, and photographic sighting – re-sighting of distinctively marked individuals, respectively, to describe community structure of spinner dolphins frequenting the nearshore island waters of the Society Archipelago, French Polynesia. First, to evaluate isolation or ‘closure’, intensive small-boat surveys at Moorea were conducted, investigating in detail the demography and genetic diversity of spinner dolphins around this island, and also taking advantage of the previous photo-identification study conducted by Poole (1995) from 1987 to 1992. Second, to address demographic and genetic connectedness, additional data (including biopsy samples and photographs) were collected around the main islands of the Society Archipelago and at Nuku Hiva in the Marquesas Archipelago, to provide insights on population structure at a larger scale (Figure 2.1b). By combining demographic and evolutionary approaches on a local and regional scale, it was hoped to provide a more comprehensive description of the long- and short-term dynamics of insular spinner dolphin populations.

Materials & Methods

Study area and small-boat surveys

From April 2002 to November 2004, spinner dolphins were photographed and genetically sampled in French Polynesia, located in the central South Pacific Ocean (Figure 2.1). Small-boat surveys were conducted (n = 189) around six islands of the Society Archipelago including Moorea, Tahiti, Huahine, Raiatea, Tahaa and Bora Bora (Figure 2.1c, Table 2.1). Efforts were made to survey the entire coastline of each island (except in Tahiti), in order to avoid geographic bias in the sampling. Because of logistical limitations, surveys in Tahiti were limited to the eastern part of the island (from Point Venus to Papara). Four boat surveys were also conducted at Nuku Hiva (n = 4), in the Marquesas Archipelago, 1,500 km north of Tahiti (Figure 2.1b). The islands of Raiatea and Tahaa were considered as one location (referred as Raiatea-Tahaa), since they are enclosed within the same lagoon. Moorea, Tahiti and Raiatea-Tahaa were visited on two consecutive years.The primary study site was Moorea, where intensive boat surveys were conducted from April to November 2002 (n = 107) and from July to September 2003 (n = 32) (Table 2.1). This island was chosen since a previous study was carried out there by Poole (1995), who conducted 275 boat surveys from 1987 to 1992, taking photographs of 249 groups of spinner dolphins. Poole (1995) also conducted 13 boat surveys along the north-east coast of Tahiti in 1988-89.

Collection and analysis of photo-identification data

During each encounter, group size was estimated by visual counts and dorsal fin photographs were taken of as many individuals as possible, regardless of distinctive marks. Photographs were taken using a digital Olympus E10 (4 megapixel CCD) equipped with a 200 mm lens and Canon Digital Rebel (6.3 megapixel CMOS) equipped with a 300 mm lens. Dorsal fin photographs were first assessed for quality independently of distinctiveness of fins. Five criteria were used to assign photographs a quality rating (Q) on a scale of 1 to 5 (poor to excellent): focus, size, exposure and percentage of the dorsal fin visible on the photo (Arnborn 1987). Only images that rated Q ≥ 3 were considered for the analyses (but see Appendix 2 for details).
Most spinner dolphins showed some unique marks on their dorsal fins but Poole (1995) found that, overall, only a limited percentage of individuals (about 15% of the population) are sufficiently distinctive to be confidently identified across time. Therefore, in this study, only dolphins with deep distinctive nicks or deformations on the edge of the dorsal fin were considered as ‘marked’ for the purpose of individual identification. This allowed comparisons of images taken from either side of an individual. This subset of dolphins is referred to as ‘Distinctively Marked Individuals’ or DMIs. All other photographed dolphins were classified as ‘unmarked’.
Based on the images of DMIs collected during the surveys, a photo-identification catalogue was created for each island. All catalogues were compared to find resights within and between islands. Inter-annual re-sightings around the same island were also recorded for islands where surveys were conducted during two consecutive years. Finally, the DMI catalogues from this study were compared to Poole’s (1995) catalogues comprising DMI photographs taken around Moorea and Tahiti between 1987 and 1992.

Biopsy sampling and DNA extraction

Skin samples for genetic analyses were collected from spinner dolphins using a small stainless-steel biopsy dart fired from a modified veterinary capture rifle equipped with a variable pressure valve (Krützen et al. 2002). Short-term behavioural responses to biopsy attempts were recorded and are reported in Appendix 3. All samples were preserved in 70% ethanol and stored at -20°C for subsequent analysis. Total cellular DNA was isolated from skin tissue by digestion with proteinase K followed by a standard phenol: chloroform extraction method (Sambrook et al. 1989) as modified for small samples by Baker et al. (1994).

mtDNA sequencing, genotyping and sex identification

An 800 base pair (bp) fragment of the 5’ end of the mtDNA control region (d-loop) was amplified using the polymerase chain reaction (PCR) and the primers lightstrand, tPro-whale M13-Dlp-1.5 (5′-TCACCCAAAGCTGRATTCTA-3′, Dalebout et al. 1998), and heavy strand, Dlp-8G (5′-GGAGTACTATGTCCTGTAACCA-3′, designed by G. Lento as reported in Dalebout et al. 2005). All amplification reactions were carried out in a total volume of 20 µL with 1 x Ampli-Taq buffer, 2.5 mM MgCl2, 0.4 µM each primer, 0.2 mM dNTPs and 0.5 U of Ampli-Taq® DNA polymerase. The PCR temperature profile was as follows: a preliminary denaturing period of 2 minutes at 94°C followed by 35 cycles of denaturation for 30 seconds at 94°C, primer annealing for 45 seconds at 55°C and polymerase extension for 40 seconds at 72°C. A final extension period of 10 minutes at 72°C was included at the end of the cycle. PCR products were purified for sequencing with ExoSAP-IT (USB) and sequenced in both directions with BigDye™ terminator chemistry v.3.1 on an ABI 3100 DNA sequencer (Applied Biosystems Inc.). Sequences were aligned using SequencherTM (version 4.1.2, Genes Codes Co.) and edited manually. Variable sites and unique haplotypes were identified using MacClade v. 4.0 (Maddison & Maddison 2000).
Samples were genotyped using 12 published microsatellite loci developed from other cetacean species (Table 2.2). Amplification via PCR was performed following standard protocols, in 10 µL volumes with 1 x Platinium-Taq buffer, 1.5 mM MgCl2, 0.4 µM each primer, 0.2 mM dNTPs and 1/8 U of Platinium-Taq® DNA polymerase, and annealing temperature varying by locus (Table 2.2). PCR products were run on an ABI 377 DNA automated sequencer with a TAMRA350 size ladder (Applied Biosystems Inc.). Data were collected by GeneScan v. 3.7, and the fragment size was measured using Genotyper v. 2.5 (Applied Biosystems Inc.). The sex of sampled dolphins was identified by amplification of a fragment of the sry gene multiplexed with ZFX positive control, as described by Gilson et al. (1998).

Table of Contents
Thesis Abstract
Dedication
Acknowledgments
Table of Contents
List of Tables
List of Figures
1. General Introduction
1.1. Overview
1.2. Brief review on the systematics of dolphins
1.3. Investigation of population structureµ
1.4. Genetic diversity
1.5. Social system
1.6. Principal methodological tools used in this study
1.7. Thesis outline and collaborators
2. Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity
2.1. Abstract
2.2. Introduction
2.3. Materials & Methods
2.4. Results
2.5. Discussion
3. Worldwide mtDNA phylogeography and diversity of pilot whale species (Globicephala spp).
3.1. Abstract
3.2. Introduction
3.3. Materials & Methods
3.4. Results
3.5. Discussion
4. Patterns of kinship and mtDNA lineage within mass strandings of longfinned pilot whales around New Zealand
4.1. Abstract
4.2. Introduction
4.3. Materials & Methods region
4.4. Results
4.5. Discussion
5. “O’ mother where art thou?” Social disruption in a mass stranding of long-finned pilot whales
5.1. Abstract
5.2. Introduction
5.3. Materials & Methods
5.4. Results
5.5. Discussion
6. Evidence of fine-scale population structure in rough-toothed dolphins from the Society Archipelago, French Polynesia
6.1. Abstract
6.2. Introduction
6.3. Materials and Methods
6.4. Results
6.5. Discussion
7. General Discussion and Future Work
7.1. Overview
7.2. Metapopulation of spinner dolphins
7.3. Pilot whales evolutionary history
7.4. Social systems and matrilineality
8. Appendices
Appendix 1
Appendix 2
Appendix 3
Appendix 4
Appendix 5
Electronic Appendices
Appendix 6
Appendix 7
Appendix 8
Appendix 9
Appendix 10
9. References

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