Stranded humpback whale ( Megaptera novaeangliae ) ( Cetacea : Balaenopteridae ) in Paraná River Delta , Buenos Aires Province , Argentina . Comments on the occurrence of marine mammals in the La Plata River Basin

The humpback whale (Megaptera novaeangliae) is distributed among most oceans and seas of the globe (except Mediterranean Sea). These whales migrate from feeding regions in the Antarctic waters to breeding areas in tropical and sub‐ tropical seas. Here we report the stranding of a female young humpback whale, which was founded dead in the vicinity of the Talavera Island, in the Paraná River Delta, Buenos Aires Province, Argentina. From the analysis of mitochondrial cytochrome c oxidase subunit I gene sequences, two novel haplotypes were found, totalizing four haplotypes described for the species. In the La Plata River Basin this species was found only twice at the end of the XIX century. Thus, the new finding constitutes an important addition to the list of cetaceans that occurs in Uruguay, Paraná and La Plata Rivers. Key-Words. Megaptera novaeangliae; Humpback whale; Delta del Río Paraná; Haplotypes; Argentina.


INTRODUCTION
The humpback whale (Megaptera novaeangliae) is a misticete cetacean that is distributed among most oceans and seas of the globe, with the exception of the Mediterranean Sea (Clapham & Mead, 1999;Clapham, 2002).These whales migrate from the feeding regions in the Antarctic Waters to breeding areas in tropical and subtropical seas (e.g., Mackintosh, 1942;Dawbin, 1966;Clapham & Mead, 1999;Clapham, 2002).In the Southern Hemisphere humpback whales have seven feedings areas (Dawbin, 1966;Clapham & Mead, 1999;Clapham, 2002;IWC, 2005).One of them, The Breeding Stock A, is distributed along the Southwestern Atlantic Ocean and constitutes one of the least known breeding groups (Andriolo et al., 2010).This stock breeds at the Brazilian coasts, at the Abrolhos Bank, where approximately 3,500 individuals were censed (Engel, 1996;Martins et al., 2001;Zerbini et al., 2006Zerbini et al., , 2011;;Andriolo et al., 2010).Genetic studies have the potential to provide data allowing to descipher long-term patterns of affiliation, breeding, and dispersal, as well as to understand important topicas on social and geographical structures of populations.In spite to that, detailed genetic studies of Humpback whales in the Southwestern Atlantic are still wanting, and the report of any evidence is urgently needed.
In the Argentine Sea the records are scarce, and this is probably because humpback whales migrate across very deep seas and avoid the large continental platform of Argentina.In this way, the strandings of the species are scarce and mostly correspond to isolated individuals (see Angeletti et al., 2014).Strandings are reported from Buenos Aires Province, Chubut Province, Tierra del Fuego Province and Islas Malvinas (e.g., Lichter & Hooper, 1984;Bastida & Rodríguez, 2009;Angeletti et al., 2014).
The aim of the present paper is to report the stranding of a female young humpback whale, which was found dead in vicinity of the Talavera Island, in the Paraná River Delta, Buenos Aires Province, Argentina.Further, we explore the genetic identification of the species and the historical revision of the occurrence of marine mammals in the La Plata River basin.

Stranding
The 21 of July of 2012 a large cetacean swimming at the Paraná River was reported by local people of Zárate town.Some days later, presumably the same individual was observed in San Pedro locality, and on July 25 th , the cetacean was found dead at the proximities of Talavera Island (33°56'00,75"S; 58°58'02,82"W).The Talavera Island is located at the 4 th section of the Delta del Paraná, at Zárate Township, emplaced at northeastern Buenos Aires Province (Fig. 1).This area is lined by a large number of watercourses, the most important being the Paraná River, that ends in the La Plata River and conforms a wide delta.

Genetic analyses
Tissue samples from the individual found in Talavera Island were collected (FHNA, hereafter).Additionally, we analyzed a sample from another individual found in Punta Alta, southern Buenos Aires province, Argentina (FHNB, hereafter).This specimen consist on skin samples and epizooic barnacles of a male specimen collected at 18/11/2011 and housed under the collection number CFA-MA-13095.This specimen was reported and its finding published in detail by Angeletti et al. (2014).Total DNA was extracted using a proteinase K digestion, extraction of proteins with phenol-chloroform method and alcohol precipitation of DNA (Sambrook et al., 1989).A fragment of 711 bp from the mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified by polymerase chain reaction (PCR) using primers FHNF 5' ATT CTC AAC CAA CCA CAA AG y FHNR 5' GTG AAA TTA TTC CGA AGC CA, specially designed for this study.Final concentrations used in PCR reaction volumes of 50 ul were: 5 μg/ml template DNA, Buffer 1X (Promega), 0.2 mM dNTPs, 0.2 μM each primer, 1.5 mM de MgCl₂ y 1.25 units of GoTaq polymerase (Promega).PCR cycling profile consisted of an initial denaturation at 94°C for 2 min, followed by thirty-five cycles of denaturation at 94°C for 1 min, anneling at 50°C for 1 min, and extension at 72°C for 1 min, and a final extension at 72°C for 5 min.PCR products were purified using a commercial kit (AccuPrep PCR Purification Kit, Bioneer) and sequenced in both direction using an ABI 337 Automated DNA Prism Sequencer (Applied Biosystems, Inc.).Sequences were aligned using CLUSTALX 2.0.11(Larkin et al., 2007) and compared with those previously published for the species (Genbank: NC006927, GQ353284, GQ353285, GQ353286, FJ590425, EU139285, EU139286, EU496287) (Table 1).From the 711 bp amplified, a 593 bp consensus region containing most variation was examined.Haplotypes were verified using DnaSP v5.10.01 (Librado & Rozas, 2009).To study patterns of geographical distribution and relationships among haplotypes, we implemented a Median-Joining  network (Bandelt et al., 1999) in PopART 1.7 (Leigh & Bryant, 2015).

RESULTS
The stranded specimen was found floating in the watercourse and resting on its right side.With the help of a backhoe and a crawler the individual was aground in the island coast to proceed with its preparation.Although its body surface was in a good state of preservation, the inner organs and its musculature were badly decomposed and its thumb was inflated by organic gas.Therefore, details of inner anatomy were not available for study (Fig. 2).
The stranded individual showed a combination of characters that unambiguously identify it as belonging to Megaptera novaeangliae: pectoral fins notably enlarged, representing ⅓ of total body length, festooned margins of pectoral fins, entirely black dorsal coloration, and head and body with a midline row of bumps, among other features (Lichter & Hooper, 1984;Bastida & Rodríguez, 2009).
The skeleton, one eye and samples of fat, skin, and muscles were housed at the Mastozoology Collection of the Fundación de Historia Natural "Félix de Azara", Buenos Aires, Argentina (CFA-MA-13084) (Fig. 3).
The individual was 10.09 meters long and was classified as a juvenile, since the length of adult specimens range from 14 to 16 meters (Clapham & Mead, 1999).Furthermore, analysis of genitalia identified it as a female (Table 1).
Five deep transverse cuts (being approximately 5 cm wide and more than 30 cm depth) were found on the left lateral side of the head of the individual, which would correspond to the impact of the propeller of a large boat on the cetacean.This certainly constitutes the cause of death of the specimen.
As other individuals of this species, its body was covered by a large number of cirripedes, specially the protuberances of the head and the anterior margin of the pectoral fins.Cirripedes were determined as Conchoderma auritum (Cirripedia, Lepadidae), an epibionthic crusta-cean frequently found in Megaptera novaeangliae (Clarke, 1966;Zullo, 1979;Félix et al., 2006;Angeletti et al., 2014).

Geographical distribution and relationships among haplotypes
From the analysis of the samples FHNA and FHNB, two novel haplotypes were found (GenBank accession numbers: KY001616 and KY001615, respectively) (Table 1).Based on the our analysis, six sequences (EU139285-6, FJ590425, GQ353284, AP006467 and NC006927) conform a common haplotype (see Appendix 1).However, since a consensus region was analyzed, mutations pres-ent beyond the consensus region length were lost and those haplotypes were grouped together (Table 2).Additionally, no phylogeographic pattern was evident based on the network results (Fig. 4).

Occurrence of cetaceans in the La Plata River basin
Humpback whales migrate notable long distances that may overcome more than 8,000 km, being probably the longest distance achieved by any mammal species (Mackintosh, 1942).Humpback whales feed in summer and spring in cold regions and during winter and autumn migrate towards breeding areas in tropical and subtropical seas (Bastida & Rodríguez, 2009;Angeletti et al., 2014).This whale is very scarce in Argentina, probably because it avoids the shallow waters of its continental platform.In fact, there has only been approximately 15 sights and strandings from 1866 to the date (see Table 3).
In the La Plata River Basin, the species has been found only twice at the end of the XIX century (Burmeister, 1867;Lahille, 1899a).Thus, this new finding constitutes an important addition to the list of cetaceans that occurs in Uruguay, Paraná and La Plata rivers.
Since the pioneering works of Burmeister at the second half of the XIX century, a large number of cetaceans were found penetrating freshwater courses of the La Plata Basin (Appendix 2).In fact, at least 19 cetacean species, and 4 pinnipeds (Carman, 2009) were found in these watercourses.These include the finding of a blue whale in the intersection between Luján and Paraná rivers by Burmeister (1872).Further, Burmeister (1867) and Lahille (1899a, b) mentioned fin whales in localities at the La Plata River coast.In addition, the holotype of the Antarctic Minke Whale, Balaenoptera bonaerensis, was found in the La Plata River, at shores of the Buenos Aires city (Burmeister, 1867;Zerbini & Castello, 2003).Minke whales are fairly common along the La Plata River coast, and the stranding of juveniles is not an uncommon fact (Marelli, 1918;Lichter & Hooper, 1984).This study/Argentina NC006927 Antarctic Note: For each haplotype, a dot indicates a nucleotide identical to the reference haplotype AP006467 at that given site.Considerable literature has been devoted to the potential causes of cetacean strandings (see Simmonds, 1997 for a review).Among the most important are the individuals that became lost during migrations, and specimens with some kind of illness, parasites or injuries (Martin et al., 1990).These specimens are in a condition in which they cannot navigate or swim properly and thus accidentally comes ashore.The same may be applied to the occasional or accidental penetration of cetaceans in some freshwater courses (see Smith & Jefferson, 2002).
However, the record of cetaceans penetrating La Plata Basin watercourses (e.g., Paraná, Uruguay, and La Plata rivers) is notably common (as explained above, at least 19 different species of cetaceans have been reported), and lost individuals are frequently found swimming in their waters.This pattern is not matched by other freshwater courses in the globe.The abnormal frequency of Cetaceans in these waters may have a special explanation.Some authors (Green, 1945in Dudok van Heel, 1962) defended the idea that some cases of strandings may be due to whales that are attempting to follow ancient migration routes through areas that have been closed by changes in sea level or sediment deposition.
In this regard, the coastal plains and surrounding areas in the south of Entre Ríos Province, together with the Paraná Delta and La Plata River have been affected by the late Pleistocene-Holocene post-glacial marine transgression (Cavallotto et al., 2005).The last evidence of this sea-level rise episode is shown as littoral deposits of mid-Holocene age, which contain concentrations rich in sea shells that are frequent in the La Plata Basin, including the La Plata River coast and southern ends of Paraná and Uruguay rivers (Guida & González, 1984;Fucks & De Francesco, 2000;Aguirre & Fucks, 2004;Martínez & Del Río, 2005).Thus, the La Plata River and the associated watercourses of southern Mesopotamia were neither an estuary nor a deltaic freshwater environment at that moment.Instead, between 6,000-6,500 and 3,000 years before present were part of a marine gulf (Cavallotto, 2002;Martínez & Del Río, 2005).Since around 3,000 years before present, there are no new records of marine influence in the area (Martínez & Del Río, 2005).
It is possible that such pre-La Plata River was an area that constituted at least a passageway for cetaceans during the Holocene.Thus, it is not improbable that cetaceans penetrating into the La Plata River are attempting to follow ancient marine routes that, since the Late Holocene to the present, are freshwater courses unsuitable for marine mammals.Although we recognize that this proposal rests on weak evidence, the unusually large number and diversity of cetaceans found in La Plata Basin may be indirect evidence sustaining this hypothesis.

Insights into Megaptera novaeangliae COI sequence analysis
Over more than a decade ago, COI region have been widely studied with the purpose to develop a "barcode" for species (e.g., Arnason et al., 2004;Amaral et al., 2007).
However, as occurs with any new genetic tool, the barcode method still presents some limitations, such as the low availability of intraspecific variation analyses in some species (e.g., Frézal & Leblois, 2008;Mitchell, 2008).In this study, we have contributed to the knowledge of the genetic diversity of.M. novaeangliae with two novel COI sequences, extending the number of described sequences from one to three for the southern distribution range of the species, and from nine to eleven for its entire geographical distribution.The lack of a phylogeographic pattern, suggested by the network results, may be due to the low number of sequences previously described for the species and analyzed in this study.In order to develop a robust genetic tool, more analyses of COI sequences are urgently needed.

Figure 1 .
Figure 1.Paraná River delta map were Megaptera novaeangliae (CFA-MA-13084) was found dead (exact location is indicated with a black dot).

Figure 4 .
Figure 4. Median-joining network based on the cytochrome c oxidase subunit I mtDNA haplotypes of Megaptera novaeangliae.Haplotypes are represented with discs and colors that indicate geographical locations.Mutational steps are indicated with stripes.

Table 1 .
Cytochrome c oxidase subunit I gene sequences analyzed and its geographic location.

Table 2 .
Polymorphic sites within 11 cytochrome c oxidase subunit I gene sequences from Megaptera novaeangliae.

Table 3 .
Sights and strandings of Humpback whales from 1866 to the date in Argentina.