Morphological and Genetics Support for a Hitherto Undescribed Spotted Cat Species (Genus Leopardus; Felidae, Carnivora) from the Southern Colombian Andes.

In 1989, a skin of a small spotted cat, from the Galeras Volcano in southern Colombia (Nariño Department), was donated to the Instituto Alexander von Humboldt (identification, ID 5857) at Villa de Leyva (Boyacá Department, Colombia). Although originally classified as Leopardus tigrinus, its distinctiveness merits a new taxonomic designation. The skin is distinct from all known L. tigrinus holotypes as well as from other Leopardus species. Analysis of the complete mitochondrial genomes from 44 felid specimens (including 18 L. tigrinus and all the current known species of the genus Leopardus), the mtND5 gene from 84 felid specimens (including 30 L. tigrinus and all the species of the genus Leopardus), and six nuclear DNA microsatellites (113 felid specimens of all the current known species of the genus Leopardus) indicate that this specimen does not belong to any previously recognized Leopardus taxon. The mtND5 gene suggests this new lineage (the Nariño cat as we name it) is a sister taxon of Leopardus colocola. The mitogenomic and nuclear DNA microsatellite analyses suggest that this new lineage is the sister taxon to a clade formed by Central American and trans-Andean L. tigrinus + (Leopardus geoffroyi + Leopardus guigna). The temporal split between the ancestor of this new possible species and the most recent ancestor within Leopardus was dated to 1.2-1.9 million years ago. We consider that this new unique lineage is a new species, and we propose the scientific name Leopardus narinensis.

Comparative mitogenome phylogeography of two anteater genera (Tamandua and Myrmecophaga; Myrmecophagidae, Xenarthra): Evidence of discrepant evolutionary traits.

The species within Xenarthra (sloths, anteaters, and armadillos) are quintessential South American mammals. Of the three groups, Vermilingua (anteaters) contains the fewest extant and paleontological species. Here, we sampled and sequenced the entire mitochondrial genomes (mitogenomes) of two Tamandua species (Tamandua tetradactyla and Tamandua mexicana) (n=74) from Central and South America, as well as Myrmecophaga tridactyla (n=41) from South America. Within Tamandua, we detected three different haplogroups. The oldest (THI) contained many specimens with the T. tetradactyla morphotype (but also several with the T. mexicana morphotype) and originated in southeastern South America (currently Uruguay) before moving towards northern South America, where the THII haplogroup originated. THII primarily contained specimens with the T. mexicana morphotype (but also several with the T. tetradactyla morphotype) and was distributed in Central America, Colombia, and Ecuador. THI and THII yielded a genetic distance of 4%. THII originated in either northern South America or "in situ" in Central America with haplogroup THIII, which consisted of ~50% T. mexicana and 50% T. tetradactyla phenotypes. THIII was mostly located in the same areas as THII, i.e., Central America, Ecuador, and Colombia, though mainly in the latter. The three haplogroups overlapped in Colombia and Ecuador. Thus, T. tetradactyla and T. mexicana were not reciprocally monophyletic. For this reason, we considered that a unique species of Tamandua likely exists, i.e., T. tetradactyla. In contrast to Tamandua, M. tridactyla did not show different morphotypes throughout its geographical range in the Neotropics. However, two very divergent genetic haplogroups (MHI and MHII), with a genetic distance of ~10%, were detected. The basal haplogroup, MHI, originated in northwestern South America, whereas the more geographically derived haplogroup, MHII, overlapped with MHI, but also expanded into central and southern South America. Thus, Tamandua migrated from south to north whereas Myrmecophaga migrated from north to south. Our results also showed that temporal mitochondrial diversification for Tamandua began during the Late Pliocene and Upper Pleistocene, but for Myrmecophaga began during the Late Miocene. Furthermore, both taxa showed elevated levels of mitochondrial genetic diversity. Tamandua showed more evidence of female population expansion than Myrmecophaga. Tamandua experienced population expansion ~0.6-0.17 million years ago (Mya), whereas Myrmecophaga showed possible population expansion ~0.3-0.2 Mya. However, both taxa experienced a conspicuous female decline in the last 10 000-20 000 years. Our results also showed little spatial genetic structure for both taxa. However, several analyses revealed higher spatial structure in Tamandua than in Myrmecophaga. Therefore, Tamandua and Myrmecophaga were not subjected to the same biogeographical, geological, or climatological events in shaping their genetic structures.

Genetics of the Andean bear (Tremarctos ornatus; Ursidae, Carnivora) in Ecuador: when the Andean Cordilleras are not an Obstacle.

One of the top carnivores in the Andean mountains is the Andean bear (Tremarctos ornatus, Ursidae), the only bear in South America. This is a flagship and key umbrella species in Ecuador because its conservation has a positive impact on the conservation of many other species in the Andes. But to preserve, first one must know the genetic characteristics of a species, among other things. For this, we analyzed six mitochondrial genes and seven nuclear DNA microsatellites of 108 Andean bear specimens sampled throughout Ecuador. We adopted three strategies for analyzing the data: by Province, by Region (north vs south), and by Cordillera. Four main results were obtained. First, the mitochondrial genetic diversity levels were elevated, but there were no differences in genetic diversity by Province or by Cordillera. By Regions, southern Ecuador had higher genetic diversity levels than to northern Ecuador. The genetic diversity for the microsatellites was only medium for the Andean bear at this country. Second, there was clear and significant evidence of female population expansions, for the overall sample, by Province, Region, and Cordillera. This population expansion was determined to have occurred in the time interval of 30,000-20,000 years ago (YA), during the last phase of the Pleistocene. We detected a population decrease to have occurred more recently, within the last 5000 years. It continued until about 300-200 YA when a population increase was again detected. Third, there were, practically, no phylogeographic pattern nor genetic differentiation among Andean bear populations in Ecuador by Province or by Cordillera for either mitochondrial or microsatellite markers. There was a little more genetic differentiation between northern and southern areas. Fourth, there was no trace of significant spatial genetic structure for the Andean bear in Ecuador in agreement with the genetic differentiation analyses. This shows that the Andean Cordilleras in this country did not present an obstacle to the dispersion of this species. Therefore, all of the Andean bear specimens in Ecuador should be treated as a unique Management Unit (MU) for conservation purposes, differently to that determined for other countries as Colombia.

Invalidation of taxa within the silvery wooly monkey (Lagothrix lagothricha poeppigii, Atelidae, Primates).

The systematics of the Humboldt's wooly monkeys (L. lagothricha; Atelidae) is essential to preserve this Neotropical primate species. Traditionally, four morphological subspecies have been described, which recently have been molecularly confirmed. However, no population genetics studies have been carried out throughout the geographical distribution of one of these subspecies, Lagothrix lagothricha poeppigii. For this reason, we analyzed nine mitochondrial genes of L. l. poeppigii mainly collected from the Ecuadorian and Peruvian Amazon in order to better understand the evolutionary history of this taxon. The mitochondrial genetic diversity levels (haplotype and nucleotide diversity) we estimated are likely the highest yet reported for L. lagothricha. Our results did not detect important genetic structure within L. l. poeppigii. Furthermore, our phylogenetic analyses did not detect any relevant molecular cluster in the area where Groves hypothesized the existence of L. poeppigii castelnaui. Therefore, based on these data, castelnaui is not a valid taxon from a molecular perspective. The most differentiated subpopulation within L. l. poeppigii was from Morona-Santiago province (Ecuador) and had a genetic distance of 0.8-1.2% relative to the other subpopulations studied. However, this genetic distance range is within the variability found within a population. We estimated the mitochondrial temporal diversification within L. l. poeppigii to have occurred during the Pleistocene, 1.8-1.2 million years ago. Similarly, all our analyses detected a strong Pleistocene female population expansion for this taxon. Diverse spatial genetic analyses, perhaps with the exception of Monmonier's Algorithm, did not detect differentiated taxa within the area analyzed for L. l. poeppigii. These genetics results could be of importance to conservation efforts to preserve this taxon as one unit.

First Molecular Phylogenetic Analysis of the Lagothrix Taxon Living in Southern Peru and Northern Bolivia: Lagothrix lagothricha tschudii (Atelidae, Primates), a New Subspecies.

We sequenced mitochondrial COI and COII genes (1,377 base pairs) of 166 woolly monkeys (Lagothrix) to determine the phylogenetic relationships of tschudii in reference to the other taxa within the genus Lagothrix, to provide the first genetic diversity level estimates for tschudii, and to reconstruct the historical demographic evolution of this taxon. The sample set included, for the first time, 10 individuals of the elusive tschudii taxon sensu Groves from southern Peru and northern Bolivia. Our phylogenetic analyses showed that these 10 exemplars formed a statistically significant and differentiated (molecularly and morphologically) monophyletic clade relative to other traditional subspecies of Lagothrix lagothricha. Therefore, tschudii should be recognized as a fifth subspecies: Lagothrix lagothricha tschudii. The temporal divergence of the ancestors of tschudii and L. l. cana was estimated to have occurred around 1.8 million years ago (MYA). Additionally, mitochondrial diversification within tschudii started no later than 0.96 MYA (Bayesian Inference) or 0.88 MYA (Median Joining -Network), respectively. In contrast to the phylogenetic trees, the FSTstatistic and the gene flow estimates showed L. l. lugens to be the least differentiated taxon of L. lagothricha from L. l. tschudii. Based on genetic distances, L. l. tschudii had the smallest average genetic distance from the other subspecies of L. lagothricha.It was also the taxon within L. lagothricha that had the smallest genetic distance from L. flavicauda. It should be related to L. l. tschudii as the first original taxon in L. lagothricha. Furthermore, the Andean mountains were extremely important in the original diversification of the Lagothrix genus and in the original diversification of L. lagothricha. Although L. l. tschudii has the smallest geographical range of all the taxa of L. lagothricha, its genetic diversity is even higher than in other taxa with wider geographical ranges, such as L. l. lagothricha and L. l. cana. L. l. tschudii showed a very slight demographic increase during the Pleistocene with a decrease of females in the last 10,000 Y, similar to that found for L. l. lugens in a previous study.

Mitogenomics phylogenetic relationships of the current sloth's genera and species (Bradypodidae and Megalonychidae).

We sequenced the complete mitogenome of 39 sloths (19 Bradypus variegatus, 4 B. tridactylus, 1 B. pygmaeus, 1 B. torquatus, 4 Choloepus didactylus, and 10 C. hoffmanni). A Bayesian tree (BI) indicated a temporal split between Bradypus and Choloepus around 31 million years ago (MYA, Oligocene) and the other major splits within each genera during the Miocene and Pliocene. A haplotype network (MJN) estimated a lower temporal split between the sloth genera (around 23.5 MYA). Both methods detected the ancestor of B. torquatus as the first to diverge within Bradypus (21 for BI and 19 MJN), followed by that of the ancestor of B. tridactylus. The split of B. pygmaeus from the common ancestor with B. variegatus was around 12 MYA (BI) or 4.3 MYA (MJN). The splits among the previous populations of B. variegatus began around 8 MYA (BI) or 3.6 MYA (MJN). The trans-Andean population was the first to diverge from the remaining cis-Andean populations of B. variegatus. The genetic differentiation of the trans-Andean B. variegatus population relative to the cis-Andean B. variegatus is similar to that found for different species of sloths. The mitogenomic analysis resolved the differentiation of C. hoffmanni from the C. didactylus individuals of the Guiana Shield. However, one C. didactylus from the Colombian Amazon specimen was inside the C. hoffmanni clade. This could be the first example of possible natural hybridization in the Amazon of both Choloepus taxa or the existence of un-differentiable phenotypes of these two species in some Amazonian areas.

Small spotted bodies with multiple specific mitochondrial DNAs: existence of diverse and differentiated tigrina lineages or species (Leopardus spp: Felidae, Mammalia) throughout Latin America.

We analysed two sets of mitochondrial (mt) DNA data from tigrinas (traditionally, Leopardus tigrinus) we sampled in Costa Rica, Venezuela, Colombia, Ecuador, Peru, Bolivia, northwestern and northeastern Argentina and southern Brazil. Additionally, the analysis included some GenBank sequences from southern, central and northeastern Brazil. The first mt set (mt ATP8+mt 16S rRNA with 41 tigrina) revealed the existence of seven different tigrina-like haplogroups. They could represent, at least, 4-6 different tigrina species following the Phylogenetic Species Concept (PSC). In the second mt set (mitogenomics with 18 tigrinas), we detected six different tigrina-like haplogroups. They could represent 4-5 different tigrina species - including a possible full new species, which has gone previously unnoticed to the world of science both morphologic and molecularly. Coat patterns of several of these different tigrinas support the molecular differences. We also detected intense hybridization in many Andean tigrina with margays (Leopardus wiedii) and ocelots (Leopardus pardalis) as well as hybridization of one Bolivian tigrina with Leopardus geoffroyi. Similar hybridization was found for many of the southern Brazilian tigrina (Leopardus guttulus). All of the temporal split estimates for these tigrina haplogroups, together with those of the Leopardus species recognized to date, began in the late Pliocene but mostly occurred during the Pleistocene. In agreement with the existence of multiple species within the traditional L. tigrinus species, we detected strong and significant spatial structure in the two mt data sets. There were clear circular clines. A major part of the analyses detected more genetic resemblance between the Central American + trans Andean Colombian and Ecuadorian tigrina (L. oncilla) with the most geographically distant tigrina from central and southern Brazil (L. guttulus; pure individuals not hybridized with L. geoffroyi). In comparison, the Andean tigrina taxa had intermediate geographical origins but were highly genetically differentiated both from the Central American + trans Andean Colombian-Ecuadorian tigrina and from the central and southern Brazilian tigrina.

Phylogeography of the Mantled Howler Monkey (Alouatta palliata; Atelidae, Primates) across Its Geographical Range by Means of Mitochondrial Genetic Analyses and New Insights about the Phylogeny of Alouatta.

We analyzed 156 specimens of diverse howler monkey taxa (Alouatta; Atelidae, Primates) for different mitochondrial genes (5,567 base pairs), with special emphasis on A. palliata and related taxa. Our results showed no relevant differences among individuals of different putative taxa, A. p. palliata, A. p. aequatorialis, A. coibensis coibensis, and A. c. trabeata. We found no spatial differences in genetic structure of A. p. palliata throughout Costa Rica, Nicaragua, and Honduras. A. p. mexicana (genetic distance: 1.6-2.1%) was the most differentiated taxon within A. palliata. Therefore, we postulate the existence of only 2 clearly defined subspecies within A. palliata (A. p. palliata and A. p. mexicana). A. palliata and A. pigra (traditionally considered a subspecies of A. palliata) are 2 clearly differentiated species as was demonstrated by Cortés-Ortiz and colleagues in 2003, with a temporal split between the 2 species around 3.6-3.7 million years ago (MYA). Our results with the Median Joining Network procedure showed that the ancestors of the cis-Andean Alouatta gave rise to the ancestors of the trans-Andean Alouatta around 6.0-6.9 MYA. As Cortés-Ortiz et al. showed, A. sara and A. macconnelli are differentiable species from A. seniculus, although the first 2 taxa were traditionally considered subspecies of A. seniculus. Our findings agree with the possibility that the ancestor of A. sara gave rise to the ancestor of A. pigra in northern South America. In turn, the ancestor of A. pigra originated the ancestor of A. palliata. Two of our results strongly support the hypothesis that the South American A. palliata (the putative A. p. aequatorialis) was the original population of this species; it has high genetic diversity and no evidence of population expansion. The Central America A. palliata is the derived population. It has low genetic diversity and there is clear evidence of population expansion. However, A. palliata and A. pigra probably migrated into Central America by 2 different routes: the Isthmus of Panama (A. palliata) and Caribbean island arch (A. pigra). Finally, the red howler monkeys from the island of Trinidad in the Caribbean Sea were not A. macconnelli (= A. s. stramineus) as Groves maintained in his influential 2001 publication on primate taxonomy. This taxon is more related to A. s. seniculus, although it formed a monophyletic clade. Future molecular and karyotypic studies will show if the Trinidad red howler monkeys should be considered as an extension of the Venezuelan taxon, A. arctoidea, as a subspecies of A. seniculus(A. s. seniculus), or, in the case of extensive chromosomal rearrangements, even a new species.

Phylogeography and spatial structure of the lowland tapir (Tapirus terrestris, Perissodactyla: Tapiridae) in South America.

We sequenced the mitochondrial cytochrome b gene of 141 lowland tapirs (Tapirus terrestris) - representing the largest geographical distribution sample of this species studied across of South America to date. We compare our new data regard to two previous works on population structure and molecular systematics of T. terrestris. Our data agree with the Thoisy et al.'s work in (1) the Northern Western Amazon basin was the area with the highest gene diversity levels in T. terrestris, being probably the area of initial diversification; (2) there was no clear association between haplogroups and specific geographical areas; (3) there were clear population decreases during the last glacial maximum for the different haplogroups detected, followed by population expansions during the Holocene; and (4) our temporal splits among different T. terrestris haplogroups coincided with the first molecular clock approach carried out by these authors (fossil calibration). Nevertheless, our study disagreed regard to other aspects of the Thoisy et al.'s claims: (1) meanwhile, they detected four relevant clades in their data, we put forward six different relevant clades; (2) the Amazon River was not a strong barrier for haplotype dispersion in T. terrestris; and (3) we found reciprocal monophyly between T. terrestris and T. pinchaque. Additionally, we sequenced 42 individuals (T. terrestris, T. pinchaque, T. bairdii, and the alleged "new species", T. kabomani) for three concatenated mitochondrial genes (Cyt-b, COI, and COII) agreeing quite well with the view of Voss et al., and against of the claims of Cozzuol et al. Tapirus kabomani should be not considered as a full species with the results obtained throughout the mitochondrial sequences.

How many genera and species of woolly monkeys (Atelidae, Platyrrhine, Primates) are there? The first molecular analysis of Lagothrix flavicauda, an endemic Peruvian primate species.

We sequenced COI and COII mitochondrial genes of 141 Neotropical woolly monkeys to provide new insights concerning their phylogeography and phylogenetic relationships. For the first time, eight individuals of the endemic and extremely rare Peruvian yellow-tailed woolly monkey (flavicauda) were sequenced at these genes and compared with other Lagothrix taxa (poeppigii, lagotricha, lugens and cana). There were four main results. (1) L. flavicauda showed a gene diversity of zero, whereas poeppigii and lugens showed high levels of gene diversity and lagotricha and cana showed more modest levels of gene diversity. The absence of gene diversity found for L. flavicauda strongly supports that it is one of the 25 more endangered primates on earth; (2) Our genetic distance and phylogenetic analyses, which included many cases of genetic introgression and recent hybridization, suggest that all woolly monkeys could be included in one unique genus, Lagotrix, divided into two species: L. flavicauda and L. lagotricha. The last species is divided into at least four subspecies. Our molecular results agree with Fooden's (1963) classification, but do not support the classification proposed by Groves (2001). (3) Poeppigii was the first taxon within L. lagotricha to experience a mitochondrial haplotype diversification, while cana and lagotricha experienced more recent mitochondrial haplotype diversification; (4) Poeppigii and lagotricha were the taxa which showed the greatest evidence of population expansions in different Pleistocene periods, whereas lugens experienced a population declination in the last 25,000 YA.