Phylogeography of the Patagonian otter Lontra provocax: adaptive divergence to marine habitat or signature of southern glacial refugia?
© Vianna et al; licensee BioMed Central Ltd. 2011
Received: 1 September 2010
Accepted: 28 February 2011
Published: 28 February 2011
A number of studies have described the extension of ice cover in western Patagonia during the Last Glacial Maximum, providing evidence of a complete cover of terrestrial habitat from 41°S to 56°S and two main refugia, one in south-eastern Tierra del Fuego and the other north of the Chiloé Island. However, recent evidence of high genetic diversity in Patagonian river species suggests the existence of aquatic refugia in this region. Here, we further test this hypothesis based on phylogeographic inferences from a semi-aquatic species that is a top predator of river and marine fauna, the huillín or Southern river otter (Lontra provocax).
We examined mtDNA sequences of the control region, ND5 and Cytochrome-b (2151 bp in total) in 75 samples of L. provocax from 21 locations in river and marine habitats. Phylogenetic analysis illustrates two main divergent clades for L. provocax in continental freshwater habitat. A highly diverse clade was represented by haplotypes from the marine habitat of the Southern Fjords and Channels (SFC) region (43°38' to 53°08'S), whereas only one of these haplotypes was paraphyletic and associated with northern river haplotypes.
Our data support the hypothesis of the persistence of L. provocax in western Patagonia, south of the ice sheet limit, during last glacial maximum (41°S latitude). This limit also corresponds to a strong environmental change, which might have spurred L. provocax differentiation between the two environments.
L. provocax has the smallest geographical range of all otter species , being distributed across the Andean-Patagonian region of southern Chile and part of Argentina [21–23]. In continental waters, the species is found from the Toltén River basin (39°S latitude) to the south of Chile . The range extends through a limited area of the Andes mountains into Nahuel Huapi National Park and Limay River in Argentina [24–26]. South of Chiloé Island in Chile (42°S) the species also occurs in marine habitats along the SFC south to 56°S . In the SFC south of Taitao Peninsula (46°S latitude), however, its distribution becomes exclusively marine [28, 27]. As L. provocax is highly dependent on the availability of crustacean prey [29, 30], the absence of the species from continental waters is related to the absence of crustaceans in the oligotrophic waters. L. provocax is solitary with intrasexual territoriality and an average home range of 11.3 km in rivers . Dispersal is limited, as shown by radio-tracked otters, where the only long-range movement was made by a juvenile male that migrated 46 km downstream after release . In freshwater, occurrence of L. provocax is dependent on crustacean distribution, which is strongly influenced by the river slope and altitude . Consequently, the distribution of L. provocax along rivers is mostly concentrated below 300 m altitude , which may limit gene flow across the Andes mountain range. As crustacean and freshwater fishes were able to live in southern Chile during the LGM, L. provocax could have had a sufficient amount of food to survive during this period. However, the occurrence of L. provocax is also dependent on riparian vegetation. Terrestrial plants such as Fitzroya cupressoides and Hypochaeris palustris were mostly absent along ice sheet cover areas during LGM [34, 35]. Thus, the question of L. provocax persistence during LGM on southern Chile likely implies a trade off between availability of food and terrestrial habitat.
So far the species has been studied mainly in the rivers and lakes at the northern limit of its distribution. Comparisons between specimens living in freshwater and marine environment are restricted to diet, and consists mainly of crustaceans such as Samastacus sp. and Aegla sp. in rivers and lakes [24, 29], shifting to marine fishes of the genus Patagonotothen sp., crustaceans  and sea urchins in the marine environment. Although otter species occur mostly in freshwater habitats, the majority of these species have also been recorded in coastal environments . Otters distributed along the coast, however, also need access to fresh water for drinking and washing their dense fur to remove accumulating salt and maintain thermo-insulation . Fresh water is abundant along the SFC and L. provocax has also been recorded in inland rivers, such as Queulat River. It is important to note that, among all otter species, only two are exclusively adapted to marine habitats, i.e. the north Pacific Ocean sea otter (Enhydra lutris) and the chungungo or south Pacific marine otter (Lontra felina). Lontra felina recently diverged from L. provocax, possibly from populations in the SFC that progressively adapted to the coastal marine habitat .
The present study analysed the phylogeographic pattern and the population structure of L. provocax, based on the mitochondrial DNA sequences from control region (CR), the NADH dehydrogenase subunit 5 (ND5) and the cytochrome b gene (Cyt-b). We aimed to infer: i) the demographic processes associated with the LGM south of the Pleistocene ice cover limit for L. provocax populations; ii) the evolutionary relationship between freshwater and marine populations; iii) the population structure within each habitat type, among different continental river basins and across the Andes mountain range, comparing L. provocax populations from Chile and Argentina.
Sampling sites of Lontra provocax analysed in this study.
Huilio River, Chile
rivers and lakes
Queule River, Chile
Mahuidanche River, Chile
Lingue River, Chile
Cua cua River, Chile
Riñihue Lake, Chile
Traful Lake, Argentina
Nahuel Huapi Lake, Argentina
Petrohue River, Chile
Darwin, Chiloé Island
Chepu River, Chiloé Island
Seno Magdalena, Magdalena Island
Valle Marta, Magdalena Island
Puyuhuapi Channel, Magdalena Island
Madre de Dios Island
Around Puerto Natales
Around Punta Arenas
D.V.D; H.IV.F; G.VI.E
The concatenated haplotype A-I-A (for CR-ND5-Cyt-b respectively) was widely distributed in the continental freshwater habitat, except in Petrohue where haplotype C-III-A was observed. Haplotypes A-I-B and B-II-A were also found only at Mahuidanchi River and Queule River respectively. A unique haplotype A-IV-I was found on Nahuel Huapi area in Argentina, not detected on Chilean rivers. All samples from rivers on Chiloé Island had a unique haplotype A-IV-A, which was not found in continental or marine sites. In the SFC, each haplotype was restricted to one or few locations.
The SAMOVA revealed increasing Φ CT values when the numbers of groups increased (K = 2-6; Φ CT = 0.55-0.72). Petrohue River was separated out in all SAMOVA partitions. The most geographically coherent partition could be defined in 4 groups: (i) most continental rivers and lakes locations including Chiloé Island (location 1 to 11), (ii) continental Petrohue river (location 9), (iii) fjords, channels and Queulat River (locations 11 to 18) and (iv) southern-most fjords and channels (locations 19 to 21). High population structure (Φ ST = 0.85, p < 0.0001) was found among 21 populations and the four groups defined. No haplotype was shared between the four groups.
Genetic diversity of mtDNA sequences for Lontra provocax.
RC+ND5+Cyt-b - Genetic Diversity
Continental rivers and lakes
0.001306 +/- 0.000791
Southern Fjords and Channels
0.7220 +/- 0.0531
0.001060 +/- 0.000663
0.8775 +/- 0.0195
0.001610 +/- 0.000922
The divergence between L. felina and L. provocax was 1.5%, whereas it reached 5.2% between L. felina and L. longicaudis, and 4.6% between L. longicaudis and L. provocax. The mean distance within the L. provocax clade was 0.3%. L. provocax mean distance between clades varied from 0.5% between a freshwater clade (B.II.A+C.III.A) and the SFC clade, 0.4% among continental freshwater clades (A.I.A.+A.I.B and B.II.A.+CI.II.A), to 0.1% among the freshwater lineages (A.I.A.+A.I.B, A.IV.A, A.IV.I, A.IV.G).
Our data evidenced a strong genetic differentiation between continental freshwater and SFC regions. The limits of the latter correspond not only to a habitat change, but also to a major biogeographic break for marine  and freshwater  species, and to the northern limit of coastal LGM ice cover [2, 3].
Southern marine fjords and channels: glacial survival?
Population displacements followed by founder effects due to recolonization of southern areas from a northern refuge would produce a signature of reduced genetic diversity, when compared to northern areas. The latter pattern was described for southern bull kelp (Durvillaea antarctica), which has reduced genetic diversity in southern Chile with a clear signature of postglacial expansion . In our case, phylogenetic reconstruction for L. provocax indicates that most haplotypes from SFC form a distinctive haplogroup emerging from a basal northern haplotype found in freshwater habitat, which could suggest a postglacial recolonization from the northern freshwater refuge. However, several results suggest a different scenario: a non-starlike network of haplotypes from SFC, an absence of historical population growth signature from the skyline plot analysis, high haplotype diversity, and highly divergent lineages. These results are not in agreement with a scenario of post-glacial expansion from northern freshwater populations. They rather support the hypothesis of a persistence of the species in this region during LGM. The hypothesis of persistence in glaciated areas is most often rejected by phylogeographic studies. Some studies have, however, shown phylogeographic results strongly supporting demographic persistence in areas supposedly covered by ice .
In Patagonia, iso-pollen lines indicate the existence of a terrestrial southern refuge  along the south-eastern coast of the Island of Tierra del Fuego, east from the Beagle Channel. The presence of a southern refuge would explain the present day plant species disjunction between Chiloé and Magallanes, and the presence of Bryophyta species and vascular herbaceous plants not distributed further north of 47-48°S . Nevertheless, the unique southern glacial refugium described for terrestrial species was not supported by our data. Single or multiple southern refugia and the persistence of the species within glaciated areas in Chilean Patagonia were debated for terrestrial [7–9, 34, 35, 41–46] and freshwater species [10–13, 47, 49]. In the case of temperate forest species, patches persisted either at the northern limits of ice cover in Chile or on the eastern limits of ice sheet in Argentinean Patagonia [34, 35, 42, 44, 48]. All these examples point to a post-glacial colonization of western Patagonia, i.e. the Chilean side of the Andes. The survival of L. provocax in non-glaciated areas of the eastern side of the Andes would have required a recolonization of the species across the Andes at multiple sites in order to allow the re-introduction of such a high genetic diversity. In addition, because the species currently occurs only in the Nahuel Huapi and Limay River area in continental Argentina [24–26], subsequent localised extinctions of L. provocax along most of the eastern refugia would be required to support such a scenario. Lastly, the unique Argentinean haplotype (A.IV.I) is derived from haplotype A.IV.A present in Chiloe Island, suggesting that its distribution was likely widespread in the recent past, and that range expansion was more likely from Chile to the eastern side of the Andes. Thus, the results do not support an origin of L. provocax diversity from eastern Patagonia.
On the other hand, glacial refugia on western coast of the Andes were suggested for some freshwater species. MtDNA sequences of the fishes Galaxias platei and G. maculatus along the western side of the Andes mountain (39°-49°S) suggest that these species survived in small southern sites due to discontinuities of the ice field [12, 49]. Also, exposed portions of the Pacific continental shelf could have constituted favourable environment for such aquatic species . Similarly, the freshwater crab Aegla alacalufi seem to have survived in glaciated areas, at least in a site identified as the El Amarillo hot springs, which was possibly left uncovered by glacial ice .
Whether multiple refugia existed or L. provocax survived all along the western southern Patagonia during LGM is still a matter of debate. The persistence of L. provocax is dependent on terrestrial habitat for dens (shelter) and on aquatic habitat for food availability. Such habitat was probably available on the eastern side of the Andes, as suggested by the extension of the distribution of Fitzroya cupressoides , allowing the survival of L. provocax in a large area and therefore allowing the persistence of high genetic diversity. In the SFC, L. provocax is known to feed not only on crustaceans and sea urchins, but also on intertidal and subtidal fishes (46% of its diet) . Ice scour can eliminate intertidal and shallow water benthos in the Southern Ocean . In the case of complete elimination of intertidal resources, diet could have been based on species such as the freshwater fish (Galaxias platei and G. maculatus), catfish (Trichomycterus areolatus) or the freshwater crab (Aegla alacalufi), which survived in the area during LGM [12, 13, 47, 49], or subtidal organisms. Otter species, such as North American river otter (Lontra candensis), the Eurasian otter (Lutra lutra) and the sea otter (Enhydra lutris), are distributed throughout extreme cold environments. L. canadensis inhabiting the marine environments in Alaska has access to two major types of prey: intertidal-demersal organisms such as fishes (Cottidae, Hexagrammidae) and crustaceans, and seasonally available schooling pelagic fishes . Similarly, L. provocax populations could have shifted their diet according to prey availability and thus persisted during the LGM in the Patagonian SFC. Although species such as the Eurasian otter (Lutra lutra) show evidence of a unique glacial refuge and low genetic diversity , other mustelids were able to survive during LGM. Gulo gulo, Mustela nivalis and Mustela erminea show adaptations for survival in Pleistocene conditions .
Southern glacial refugia or adaptation to marine habitat
Our data show a monophyletic haplogroup for most haplotypes from the SFC range of L. provocax (43°S to 53°S), distinct from freshwater habitat haplotypes. Such differences between the L. provocax populations inhabiting the freshwater and marine environments suggest either past genetic isolation, and/or restricted gene flow between them at the present time, allowing genetic drift or natural selection to operate. Changes in the L. provocax diet and a greater ability to swim larger distances as required in the SFC could eventually lead to adaptation to the marine environment, if plasticity is limited. However, patterns of genetic diversity generated by gene surfing during recolonization are similar to those generated by selection and could thus be mistakenly interpreted as adaptive events . Similarly to selection and unlike most other demographic effects, gene surfing generally does not affect all loci, and thus seems especially difficult to distinguish from directional selection . All otter species, except the sea otter (Enhydra lutris) and the chungungo (Lontra felina), are dependent of freshwater habitat . Nevertheless, freshwater sources are abundant in SFC and L. provocax can temporarily return to rivers to access a supply of fresh water. Moreover, all but four freshwater otter species have also been recorded along the coast  (Lontra canadensis, Lutra lutra and Lontra longicaudis, among others). Nevertheless, no genetic surveys have been conducted to determine divergence of otter lineages from different environments. Lontra phylogeny based on mtDNA markers revealed the recent divergence between L. provocax and L. felina about 883,000 years ago (95% HPD: 0.16-1.89 mya) with a possible speciation of L. felina from L. provocax living on SFC . This speciation scenario is in agreement with the adaptation hypothesis of L. provocax to the marine habitat in SFC.
Although higher haplotype diversity was expected along northern populations due to the persistence of rivers and forests, low haplotype diversity (compared to SFC) but high divergence among haplotypes was observed. Our results are concordant with the hypothesis of a recent loss of genetic diversity in freshwater environments due to hunting and habitat destruction. This is specifically supported by: i) A.I.A haplotype shared among several locations; ii) two highly divergent clades; iii) two divergent haplotypes (A.I.A and B.II.A) in the Queule River. Genetic theory predicts that during population bottlenecks low frequency alleles are lost by genetic drift . Similarly, L. provocax intermediate haplotypes are not seen on the MJN, suggesting that they were eliminated within the freshwater range. This pattern is consistent with the history of the L. provocax populations in the region. Indeed, L. provocax populations have been eliminated from the north of its past range. The northern limit of distribution changed from Cauquenes and Cachapoal rivers (34°S) to Tolten River basin (39°S) . Its small geographical range has been strongly impacted by anthropogenic activities resulting in a decline to less than 10% of its former distribution in freshwater habitats. L. provocax was intensively hunted for its fur; and hunting continued until the 1970's in some southern localities . Furthermore, the species activity has been significantly reduced in areas where riparian vegetation was removed or watercourses were disturbed or recently polluted by pulp factories [57, 58]. Riparian vegetation significantly influences the presence of crustaceans and consequently the occurrence of L. provocax in the area [30, 32, 57].
The reduction in the distribution of L. provocax led to the classification of the species in Chile as ''endangered'' in the northern range between the O'Higgins and Los Lagos regions, in rivers and lakes, and ''insufficiently known'' for the Aysén and Magallanes regions, where distribution was largely marine . Although L. provocax in freshwater habitats mostly occur below 300 m altitude , the majority of National Parks and Reserves (> 90%) in south-central Chile (35.6° to 41.3°S) are located above 600 m altitude , and therefore do not serve the conservation of this species.
Our data shows that the survival of the species along SFC during glacial cycles maintained a high diversity along SFC. Large-scale temperate deforestation in Chile has progressed from north to south . Human populations are concentrated in the central-south region of Chile, and are less dense south of Chiloé Island. Thus, southern L. provocax populations have not been greatly impacted by anthropogenic actions, and have maintained high genetic diversity compared to northern freshwater populations. L. provocax populations along SFC are, however, barely studied, and increasing human activities in this area are a potential threat to these populations.
Our results evidenced the persistence of a semi-aquatic carnivore species, the huillín, in western Patagonia along areas covered by ice sheet during LGM. Marine habitat of the SFC played an important role for L. provocax survival during LGM, probably associated to the survival of other freshwater and marine species that may have represented a persistent food source for the huillín. Therefore, genetic differentiation between northern exclusively freshwater habitat dominated by riparian vegetation and SFC may be explained by some ecological differentiation between both kinds of habitats. This is an interesting clue for understanding why so many aquatic species seem to have persisted in glaciated areas.
Study area, sample collection and DNA extraction
A total of 57 feces and 18 tissue samples were collected. These included blood from captured animals, muscle from carcasses of animals that died of natural causes, and pelts confiscated by authorities due to illegal hunting. Feces and muscle tissue were preserved in pure ethanol. The sampling range included the full range of the species (Figure 1) from the northern limit in Chile, the Huilio River tributary of Tolten river (38°58'S) to southern Patagonia (53°08'S, Table 1, Figure 1, 2). In addition we sample the only known population of the eastern side of the Andes: the Nahuel Huapi Area. A total of 21 locations were sampled, nine in continental rivers and lakes (CRL), two in rivers from Chiloé Island (CI) and ten marine locations in the fjords and channels (SFC).
Mitochondrial DNA control region (CR), NADH dehydrogenase subunit 5 (ND5) gen and cythocrome b (Cyt-b) gen were amplified using primers described by : LfCR-2F and LfCR-1R; ND5-DF1 and LfND5-R; and LfCYTB-1F; LfCYTB-1F; LfCYTB-2F and LfCYTB-2R.
PCR reactions were carried out following . PCR amplification was confirmed by electrophoresis of products with ethidium bromide in 0.8% agarose gels and visualization under UV light. Amplicons were purified using QIAquick PCR purification kit (Qiagen) and sequenced using amplification primers by Macrogen Inc., Seoul, Korea. All sequences have been deposited in the GenBank [GenBank: GQ843803-GQ843824, and HM997011-HM997017].
The sequences were aligned and mutations were confirmed by eye according to the chromatogram using Proseq ver. 2.91 . All sequences were aligned and haplotypes were identified using ClustalX ver. 1.83 .
Spatial analysis of molecular variance (SAMOVA) was implemented by SAMOVA ver. 1.0  to define groups based on the geographic distribution of the genetic diversity. SAMOVA were performed for 21 locations testing from 2 to 7 groups, each of which with 100 initial conditions. The groups of populations geographically homogeneous are defined by maximizing Φ CT values (among group variance) and minimizing Φ SC values (among populations within group variance). Haplotype (h) and nucleotide diversity (π) were calculated using Arlequin program ver. 3.0  for all data set and for the different environments.
Deviations from a neutral Wright-Fisher model were performed by calculating Tajima's D and Fu's Fs statistics [65, 66]. We tested the demographic  and spatial expansion  models by calculating the sum of squared differences (SSD) between the observed and an estimated mismatch distribution obtained by 1,000 bootstrap. The P-value of the SSD statistic was calculated as the proportion of simulated cases that show a SSD value distinctive from the original. Calculations were performed in ARLEQUIN, using 1,000 bootstrap to evaluate significance. To estimate the shape of population growth through time for the individuals distributed along the ice sheet coverage area during the LGM, we constructed Bayesian skyline plots implemented in BEAST v 1.4.8 . The appropriate model of nucleotide substitution was HKY+I determined using ModelTest ver. 3.06 . Five million iterations were performed, of which the model parameters were sampled every 1000 iterations. Throughout our analysis, we assumed a within-lineage per site mutation rate of 6%Ma. Demographic plots for each analysis were visualized using Tracer v1.0.1 .
We applied the Partition Homogeineity test (10,000 permutation) to assess the congruence of the evolution rates among CR, ND5 and Cytb using PAUP ver. 4.0b8 . The evolutionary relationship among concatenated CR+ND5+Cyt-b haplotypes was investigated by a Median Joining Network using Network ver. 18.104.22.168 , and phylogenetic reconstructions based on Bayesian (BA) methods. Four divergent haplotypes of Lontra felina , were incorporated in the phylogenetic reconstruction, whereas a L. longicaudis haplotype from the Amazon (this manuscript) was used as an outgroup. The substitution model of DNA evolution was selected based on AIC using Modeltest ver. 3.06 . BA was performed by MrBayes ver. 3.1.2  using the general type of the best fit model parameters defined for the data set, in which four independent analyses were run with four chains each, for six million generations and then sampled at intervals of 1,000 generations. The first 25% of sampled trees were discarded to ensure stabilization and the remaining used to compute a consensus tree. The split frequency was below 0.004, confirming that sampling was from the posterior probability distribution. Mean distance between clades and species was calculated using Mega v.3.1  using p-distance.
This work was supported by Universidad Andrés Bello-DI-06-06/R, Rufford Small Grant for Nature Conservation, Earthwatch Institute and FONDECYT 1100139. Vianna was supported by a CONICYT Doctoral Fellowship, CONICYT Thesis Project AT-23070034.. Special thanks to René Monsalves, Attia Zerega, Juan Carlos Marín, Gerardo Porro, Carla Pozzi, Javier Lucotti who helped with sample collection. Samples from southern Patagonia were collected by Servicio Agricola y Ganadero (SAG) after illegal hunting. Florance Tellier, Andrés Parada and Emma Newcombe helped with analysis and English. All Chilean samples were collected according to permits: Subsecretaria de Pesca (686-2006; 1588-2009; 1228-2009).
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