Phylogeographic description of Steno bredanensis and Physeter macrocephalus, inferred from the analyses of mitochondrial DNA of stranded individuals in the Caribbean Sea

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Phylogeographic description of

Steno bredanensis

and

Physeter

macrocephalus

, inferred from the analyses of mitochondrial DNA of

stranded individuals in the Caribbean Sea.

Ramiro Rueda; Susana Caballero PhD

_________________________________________________________________________________________________

1. Introduction

Cetaceans, in general, are really dynamic animals that are constantly moving from one site to another (Moura et al. 2014; Parsons et al. 2013). As an example of this, many of them migrate depending on the season of the year, the water levels, the temperature and some other biotic and abiotic factors (Hoelzel, 1991). Although there are many species that present a particular fidelity to an specific home range, these marine mammals are commonly involved at least in reproduction and foraging spot changes that lead groups or individuals to spread their genes and increase genetic variability (Mesnick, 2011). Rough-toothed dolphins (Steno bredanensis) and sperm whales (Physeter macrocephalus) are considered cosmopolitan marine mammals in terms of distribution. While it is true that Physeter specimens are found in a considerably wider range in comparison with rough-toothed dolphins, both species could be found almost everywhere in tropical areas (Taylor et al, 2008). For both, S.bredanensis and P. macrocephalus, conservation status is not as startling as it is for other cetacean species and their populations seem to still be plentiful.

Steno bredanensis is a least concern classified specie according to the IUCN red list. However, unlike other dolphin species including, inter alia, Tursiops truncatus and Sotalia guianensis, rough-toothed dolphins have primarily an offshore distribution that limits the development of scientific studies. Therefore little is known about this specie and the available information is solely based on stranded individuals and a few sightings (Kiani, Iqbal, Siddiqui and Moazzam, 2013). Considering this, it has

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been estimated that its global abundance consists of approximate 200,000 individuals, being the eastern tropical pacific a hot spot with about 145,000 of them. Rough-toothed dolphins have also been spoted in deep oceanic waters in the Atlantic, Pacific and Indian oceans and in the Mediterranean and Caribbean seas. (Perrin and Walker 1975; Leatherwood and Reeves 1983; Reeves et al. 2003; Gannier and West 2005). Due to this dolphin’s interoceanic dispersal, it becomes interesting to asses the genetic links between geographically distant populations in order to determine possible explanations that led to this specific spatial arrangement patterns. Amaral et.al, 2012 studied genus delphinus and determined that past climatic changes influenced population structure and demography suggesting that common dolphins had recurrent demographic expansions concomitant with changes in sea surface temperature during the Pleistocene (Amaral et al. 2012). Probably, this could be the strongest argument to support S.bredanensis´s distribution although other reasons may not be discarded.

On the other hand, Physeter macrocephalus´s distribution reaches even polar waters, however it is well known that the worldwide population of sperm whales has been greatly reduced since whaling became a common practice, during the last 40 years the global abundance has been reduced by more than 60% with a current estimate of 360,000 individuals (Whitehead, 2002). These massive historical hunts have principally undermined the male-to-female ratio reducing birth rate (Whitehead, 2002). Along these lines, the large toothed whale is considered a keystone specie in many deep ocean ecosystems (Rendell, 2014) and, as expected, many conservation campaigns have triggered the development of scientific research. Added to the great importance in terms of conservation that this kind of study means, it is also interesting to highlight that sperm whales, as many other cetacean populations, present a matrifocal social behavior, in reference to the formation of units composed by females and calfs. Various authors, such as Whitehead et.al 1998, have also proposed philopatry among female toothed whales suggesting population structuring defined to specific oceanic regions. All of these aspects make this specie

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an interesting model to establish genetic differences among populations from distant ocean basins (Engelhaupt et al. 2009).

In order to have an insight on the phylogeography of these species, application of molecular markers are an effective tool. That being the case, mitochondrial genes are used for determining systematics and phylogenetics of cetaceans, mainly because of its elevated mutation rate, stability of genetic content, homogeneous types of mtDNA within individuals and matrilineal inheritance (Dillon, 1993). Therefore, the purpose of this study is to perform a genetic analyses in order to evaluate phylogeographic patterns of sperm whales and rough toothed dolphins from the Caribbean with population around the world. These analyses were done by amplification of two mitochondrial DNA regions corresponding to the citochrome b and control region.

2. Materials and methods

2.1 Sample collection

2.1.1 Physeter macrocephalus

Tissue samples were taken from two stranded sperm whales, refrigerated at -20°C and preserved in 70% ethanol. The samples were obtained at two different locations in the Colombian Caribbean Sea, one at Santa Marta city in the department of Magdalena and the other one at the Urabá gulf in Turbo, located near to the º1 border with Panamá. Additionally, 16 previously published sequences, in GenBank, were used to find similar haplotypes between Colombian sperm whales and some other populations reported from the North Atlantic, Northwest Atlantic, South Pacific basins and the Mediterranean Sea and the Gulf of Mexico(see Table 2).

2.1.2 Steno bredanensis

The blood, bones and tissue samples were also collected from stranded dolphins. Tissue was preserved in 70% ethanol while blood was preserved in EDTA. The dolphins used for this project were found at the Colombian Caribbean sea (n=1) and

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Puerto Rico (n= 4), the rest of the sequences (n=22) were previously reported from the Eastern Pacific, South Pacific, Atlantic and Indian ocean basins. (see Tables 3 and 4) .

2.2 DNA extraction, amplification and sequencing

Total genomic DNA from all samples was extracted using the DNeasy blood and tissue kit (Qiagen).

2.2.1 Physeter macrocephalus.

Extraction products were subsequently amplified using a conventional PCR protocol, employing specific primers that annealed in the posterior and anterior extremes of the control region. The mtDNA amplicons were cleaned applying the GeneJET PCR purification KIT by Thermo Scientific.

2.2.2 Steno bredanensis.

The amplification was done following a standard PCR protocol and the conditions of Caballero et.al 2008. Mitochondrial DNA control region and Cytochrome b PCR products were purified using the GeneJET PCR purification KIT by Thermo Scientific.

All PCR products were secuenced using the Sanger method on an ABI 3500 at Universidad de los Andes.

2.3 Data analyses

All the obtained sequences were alligned and edited using Geneious R7 7.1.4 and the haplotypes were determined with MacClade. For the Steno bredanensis samples, phylogeographic comparissons consisted of approximately 450bp consesus sequences for cytb and 420 bp for control region. These sequences were later compared with 5 cytb sequences and 18 control region sequences which were all gathered from GenBank (see Tables 2, 3 and 4). A consensus sequence of 399 bp of the control region was analysed for the two P.macrocephalus samples. These

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sequences were later compared with sequences downloaded from GenBank (see Table 2) . Haplotype networks were constructed for each specie and it respective mtDNA region, all using TCS 1.21. . In addition, phylogenetic trees were built to describe relationships between the established haplotypes and outgroups (Tursiups truncatus and Phocoena phocoena for dolphins and Balaena mysticetus for the sperm whales), to do so the Bayessian tree method implemented in BEAST v2.1.3. was used. The output tree file obtained was visualized with FigTree v1.3.1. Finally, analyses of molecular variance (AMOVA) were calculated using Arlequin 311. Nucleotide and haplotype diversity were estimated and the genetic differences between the previously defined populations were obtained for each species, nevertheless due to sample sizes the results were not significant so this was not taken into account for the analyses.

3. Results

A total of 9 sequences were obtained for the 7 individuals included in the study. Only the sequences with a high identity percentage were analyzed in order to reduce the bias of sequencing process. From these 9 sequences, 2 correspond to control region sequences of the stranded individuals of the Physeter macrocephalus samples collected in the Colombian Caribbean, 4 to cytb region of Steno bredanensis of the Puerto Rican Caribbean and the remaining 5 sequences belong to control region of Steno bredanensis samples of Colombian and Puerto Rican Caribbean individuals. Taking into account the specie and the mtDNA region it corresponded to, and including the sequences obtained from GenBank, the haplotypes were determined.

For S.bredanensis cytb, 9 sequences in total were analyzed allowing the differentiation of 4 haplotypes. Every haplotype was shared by a pair of individuals with the exception of HapC, which was shared by 4 individuals, and HapA, which was unique. The haplotypes were distributed throughout the different oceanic

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basins which included: South Atlantic, Eastern Tropical Pacific, North Atlantic, Puerto Rican Caribbean, Colombian Caribbean, Indian and North Pacific. Additionally, HapC was the only haplotype identified in the four of the oceanic basins, meanwhile three of the four haplotypes were found only in the Puerto Rican Caribbean (see Fig 5).

For S. bredanensis control region, a total of 23 sequences including sequences obtained from GenBank, were used for the analysis. From these, 21 haplotypes were defined which were located in South Atlantic, Eastern Tropical Pacific, North Atlantic, Puerto Rican Caribbean, Colombian Caribbean, Indian and North Pacific ocean basins. In this case, the haplotypes were unique and more restricted to specific basins. Only the haplotype AP12 was identified in two different locations and shared by two different individuals. Likewise, two distinct individuals also shared AP7, but here it was only present in a single location (Fig 3). On the other hand for P. macrocephalus 18 sequences were analyzed. Among these, 7 haplotypes were defined which were present in South Pacific, Northwest Atlantic, North Atlantic oceans and Colombian Caribbean, basins as well as in the Gulf of Mexico. Of the determined haplotypes, only H1 was shared by more than one individual and was present in 3 different oceanic basins. The other haplotypes were restricted to a single location aside from H6 and H4, which were both present in the Northwest Atlantic Ocean (see Fig 1 and Table 2).

Phylogenetic trees for S.bredanensis and P. macrocepahlus were constructed using the sequences of all individuals. The trees evidenced the relation between the haplotypes defined, where some individuals sharing the same haplotype were together in the same clade.

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P. macrocephalus control region

Fig 1. Parsimony network of Physeter macrocephalus mtDNA control region haplotypes. White circles in branches represent missing or extinct haplotypes and the numbers represent the site changes.

Fig 2. P. macrocephalus Individual´s tree phylogeny of mitochondrial data sets estimated with *Beast method.

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Steno bredanensis control region

Fig 3. Parsimony network of Steno bredanensis mtDNA control region haplotypes. Small white dots represent missing haplotypes and the numbers on the branches represent site changes.

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Fig 4. S.bredanensis control region individual´s tree phylogeny of mitochondrial data sets estimated

with *Beast method.

S.bredanensis Cytb

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Fig 6. S.bredanensisCytbindividual´s tree phylogeny of mitochondrial data sets estimated with *Beast method.

Fig 7. Map showing possible S.bredanensis migration routes impulse by climatic changes and the oscillating water temperatures taking into account routes defined by Amaral et al. 2012 (Red color represents warmer water currents).

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4. Discussion

4.1 P. macrocephalus:

As it is shown in the network (Fig 1), seven different haplotypes were obtained from sperm whales sequence alignments (See also table 2 from Appendix). Haplotype 1 (H1) becomes the analyses focus of attention mainly because it presents a really interesting distribution, so that H1 appears to be present in geographical distant locations such as it is the Colombian Caribbean coasts from the Mediterranean Sea and the South Pacific

Ocean. In general, results showed by the tree (Fig 2) and the network are interesting. In the

case of haplotype number 1, as expected, almost all individuals belong to the same geographical location, the Mediterranean Sea. Anyhow, there are some strange cases that wide this discussion, for example two different samples that corresponds to the Colombian Caribbean (Pmacura and Pmacstm) and one that belongs to the South Pacific Ocean (Pcatcr) are still being classified as H1 by the haplotypes network. Likewise, network results suggests that each location studied, presents its own haplotype being this information consistent taking into account that the females of sperm whales are phylopatric animals (Engelhaupt et al, 2009; Mesnick et al, 2011). Also, contrasting the network results with the tree it is possible to see that H1, the ancestral haplotype according to the network, is conforming a monofiletic group shared with two other invididuals that belong respectively to H5 and H4. However, referring to the haplotype network it is shown that H5 and H4 are relatively closely linked with H1.

As expected with cetacean species, translating geographically distant individuals into genetically tied populations is a complex task due to the fact that species with large populations and widespread distributions are asocciated with high mitochondrial DNA diversity (Mulligan et al. 2006). Nevertheless, sperm whales are known to be an exception because even though they exhibit large populations and wide ditribution ranges their mitochondrial diversity is particularly low (Alexander et al. 2013). Based on this, our results evidenced these findings because in our samples distant individuals

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were closely related. There are some other reasons that could explain the apparent tight relations between this vastly separated samples. Thus, sperm whales are known to have matrifocal behavior, which means that females remain in a reduced spatial range with their calfs while mature males migrate long distances. According to this and taking into account that mtDNA is strictlly inherited from females, which are known to present site fidelity, the related samples could not be surely associated to different distant linked populations and due to the restricted number of samples it is not possible to stablish a population struture that allows the determination of the location of origin. What could be a possible explanation, is that the suggested connection between this distinct sites might be accounted to migrant males belonging to a central settled population that is not necessarily close to the stranding site.

4.2 S.bredanensis:

As mentioned before, Steno bredanensis is one of the least studied cetacean species. Therefore, phylogeographic and population genetic studies are unavailable and its phylogenetic relation to other cetaceans is still of great discussion between authors, as well as its ecological behavior and distribution. The vast majority of the data obtained for this specie comes from ashore individuals and occasional records of sightings; reason why conclusions based on evidence of distribution behavior are limited and hence primarily based on studies of other cetacean species.

The results gathered for S.bredanensis haplotype analysis were interesting. For cytb, 4 haplotypes were defined. Of these, HapC was shared by 4 individuals suggesting an interoceanic dispersal. On the other hand HapA, the ancestral haplotype suggested by the network (Fig 5) was unique and located in the Pacific Ocean, which has been previously identified as a marine center of evolutionary origin (Amaral et al, 2012; Briggs, 2003; Cunha, 2011; Tougard, 2001). When compared with the phylogenetic tree (Fig 6) the relation between the individuals and the haplotype was almost perfect, each clade matched each of the haplotypes and the shared individuals were closely branched. Thus, individuals from close geographic locations were also defined by the tree as having closely related haplotypes. This confirms our findings, and proposes an

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evolutionary connection between the interoceanic populations, and their ancestral origin being the Pacific Ocean. HapA was the haplotype that showed more relations to the other ones, reason why it was established as the ancestral haplotype when designing the network. On the other hand, HapB and HapD presented a more limited distribution near in the Atlantic Ocean and indicating a more recent evolutionary appearance.

A series of hypothesis could be proposed to explain the interoceanic distribution of HapC, based on biographic scenario and vicariance advised for other delphinidae species. Amaral et al 2012 studied common dolphin’s influences of past climatic changes on historical population structure and demography. She proposed two scenarios regarding species of the Delphinus genus, which present various interoceanic populations in accordance with the results described for the rough-toothed dolphin. The accurate scenario, which accounted for the origin of common dolphin´s and may also explain S.bredanensis spreading, suggests that climatic and oceanography changes in the Pleistocene are the principal triggers. Glaciations during this geologic era were characterized by extremely low temperatures and freezing, mainly in the north pole of the planet. Due to this historical climatic events, few warm waters were significantly displaced to the south carrying with them terrestrial and marine wildlife (Tougard, 2001). However in our specific case our samples are not enough to confirm that HapC´s distribution was affected by either of these hypotheses.

An unequal frequency and distribution of haplotypes between Atlantic and Pacific oceans evidenced the identification of the Pacific as the site of ancestral populations, which matches the results found for the rough-toothed dolphin fixed by the time divergence tree built in BEAST. The dispersal to the Atlantic Ocean may be pressured by cooling events of the tropical Pacific during the Pleistocene period, which had various consequences including disturbances in sea levels, water temperatures, oceanic current anomalies and shoreline configurations are known to have also impacted other marine organisms (Amaral et al. 2012, Taguchi et al. 2010; Liu et al 2011). For the case of dolphins, it seems the dispersion was from the Pacific Ocean to the Indian Ocean, and subsequently to the Atlantic Ocean (Fig 7). The dispersal from

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the Indian Ocean to the Atlantic may have been facilitated by the Agulhas current system which projects warm water masses to the west passing through South Africa (Amaral et al 2012; Peeters et al 2004). This is supported by the haplotype network were it is possible to identify haplotypes of the Pacific nested within the Atlantic, as it was also mentioned for common dolphins by Amaral et al 2012.

In spite of Amaral’s distribution hypothesis, that analysis was not possible for this study mainly because defined populations were based on a small number of individuals so it did not facilitate the estimation of population sizes. It becomes evident that more data must be gathered to conclude on this respect therefore the hypothesis here presented should be posteriorly validated or refuted.

5. Conclusions

In conclusion, molecular mtDNA analysis is confirmed to be a very useful tool for studying evolutionary history of species. Nevertheless a large number of samples must be gathered in order to reduce statistical bias and allow the construction of a genetic population model that can be extrapolated to real situations for the determination of site origins and distribution behaviors that aid in the development of conservation strategies. We encountered a several number of limitations that must be taken into account in subsequent investigations in order to improve the significance of the results. It is crucial to have more information regarding the samples used, like sex and geographical coordinates, this way we can understand exactly form where is the information coming from. In the case of P. macrocephalus, knowing the samples’ sex would have allowed an appropriate explanation of the interoceanic haplotypes, taking into account that these species are matrifocal and large distance migrations are only observed in mature males. Also, highlighting that samples were obtained from stranded individuals, we are not sure if the place where they were found corresponds to an accurate location in which the animals used to be or if it is just product of the oceanic dynamics and currents. Likewise, the reduced number of studies available for S. bredanensis restrain result analysis therefore it is not possible to confirm a hypothesis based on the data used. The results obtained are a preliminary view of

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biogeographic scenarios that may explain S. bredanensis and P.macrocephalus distribution patterns. For S.bredanensis there are no previous studies regarding its phyleogeography so the identified haplotype patterns are a preview of this cetacean species’ dispersal, which may also be influenced by climatic changes in ancestral periods as it has been confirmed for other marine organisms.

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6. Appendix

Table 1. Collected samples´s information.

Table 2. P.macrocephalus GenBank sequences’ information with corresponding haplotypes.

Specie Code Date Location

P.macrocephalus pmacstm March,2014 Colombian Caribbean (St Marta)

P.macrocephalus pmacura * Colombian Caribbean (Urabá)

S.bredanensis Sbreprtru/Sbreprun 31/04/08 Northeast Caribbean (Puerto Rico)

S.bredanensis S.bre.7JULY1603 16/07/2003 Northeast Caribbean (Puerto Rico)

S.bredanensis Sbrepra 03/04/2008 Northeast Caribbean (Puerto Rico)

S.bredanensis Sbrepren 25/01/2008 Northeast Caribbean (Puerto Rico)

S.bredanensis Sbregaira 22/04/2012 Colombian Caribbean (Gaira)

GB Accession # Tree code Location Haplotype

NA pmacstm Colombian Caribbean (SM) H1

NA pmacura Colombian Caribbean (Ur) H1

X72203.1 pmaccre North Atlantic H2

AY582747.1 pcatcr South Pacific H1

AY822115.1 pcathsp North Pacific H3

DQ512934.1 pcathn Northwest Atlantic H4

KF233572.1 pcatnu Mediterranean Sea H1

KF233571.1 pcatoch Mediterranean Sea H1

KF233570.1 pcatsiet Mediterranean Sea H1

KF233569.1 pcatseis Mediterranean Sea H1

KF233568.1 pcatcinc Mediterranean Sea H1

KF233567.1 pcatcuat Mediterranean Sea H1

KF233566.1 pcattres Mediterranean Sea H1

KF233565.1 pcatdos Mediterranean Sea H5

KF233564.1 pcatuno Mediterranean Sea H1

DQ512948.1 pcathbb Northwest Atlantic H6

DQ512945.1 pcathy Gulf of Mexico H7

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Table 3. S.bredanensis control regionGenBank sequences’ information with corresponding haplotypes.

GB Accession # Tree code Location Haplotype

EF027032.1 Sbreznu Atlantic Ocean HapC

AF084076.1 Sbreafss Eastern Tropical Pacific Ocean HapA

AF084077.1 Sbreafst North Atlantic Ocean HapD

NA Sbrepra Puerto Rican Caribbean HapD

GQ253567.1 Sbrekar Indian Ocean HapC

EU121107.1 SbreCRS138 North Pacific Ocean HapC

NA Sbregaira Colombian Caribbean HapB

NA Sbrepren Puerto Rican Caribbean HapB

NA Sbreprun Puerto Rican Caribbean HapC

Table 4. S.bredanensis cyt bGenBank sequences’ information with corresponding haplotypes.

GB Accession # Tree code Location Haplotype

AB610364.1 SbreEW04730 Eastern Pacific Ocean AP8

JQ798157.1 Sbreh7 South Pacific Ocean AP9

JQ798148.1 Sbre02Mo03 South Pacific Ocean AP16

JQ798149.1 Sbre03Hu12 South Pacific Ocean AP7

EU121131.1 SbreCRSbrez138 North Pacific Ocean AP6

EF027007.1 Sbreznu Atlantic Ocean AP4

AY842471.1 SbreMQ137 Atlantic Ocean AP17

FJ411044.1 Sbrekar Indian Ocean AP12

NA Sbreprtru/Sbreprun Puerto Rican Caribbean AP5

NA Sbrepra Puerto Rican Caribbean AP18

NA Sbregaira Colombian Caribbean AP21

NA Sbrepren Puerto Rican Caribbean AP20

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