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.

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.

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.

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.

Genetic characterization and structure of the endemic Colombian silvery brown bare-face tamarin, Saguinus leucopus (Callitrichinae, Cebidae, Primates).

We analyzed 115 Saguinus leucopus, from four Colombian departments (Antioquia, Bolivar, Caldas and Tolima ), for 701 bp of the mt COII gene and at 10 microsatellite loci to estimate gene diversity levels, possible molecular subspecies and historical demographic changes in this species. This endemic Colombian species showed an elevated gene diversity in this gene, although its geographical distribution is very restrictive and extremely threatened by habitat fragmentation. The mt COII gene did not show any geographical structure in the distribution of the haplotypes within this species, but it did show a noteworthy population expansion throughout the history of this species. A Bayesian analysis showed that the haplotype diversification of this species began around 1.6 million years ago (MYA), whilst a haplotype network gave the beginning of this diversification at around 0.5-0.6 MYA. Forty-seven individuals out of the 115 were analyzed for 10 DNA microsatellites. The genetic diversity was relatively elevated for this kind of marker too, and comparable to that found in other Neotropical monkeys with a wider geographical distribution. Two gene pools were detected with the microsatellites, one in the northern distribution area (Antioquia) and the other in the southern distribution area (Tolima). No tests detected any bottleneck affecting this population; however, two procedures (k test and Kimmel et al. 1998 test) detected significant population expansion for the microsatellite markers, like that seen with the mt COII gene.

Molecular relationships and classification of several tufted capuchin lineages (Cebus apella, Cebus xanthosternos and Cebus nigritus, Cebidae), by means of mitochondrial cytochrome oxidase II gene sequences.

The morphological systematics of the tufted capuchins is confusing. In an attempt to clarify the complex systematics and phylogeography of this taxon, we provide a first molecular analysis. We obtained mitochondrial cytochrome oxidase II (mtCOII) gene sequences from 49 tufted capuchins that had exact geographic origins from diverse lineages in Colombia, Peru, Bolivia, French Guyana, Brazil, Argentina and Paraguay and that belonged to clearly recognized morphological taxa. This project had 4 main findings: (1) we determined 2 established and related taxa in the northern Amazon River area, which we named C. a. apella and C. a. fatuellus. C. a. apella is distributed from French Guyana until, at least, the Negro River in the northern Brazilian Amazon, whereas C. a. fatuellus is distributed throughout the Colombian Eastern Llanos and the northern Colombian Amazon. We also determined 2 other southern C. apella taxa, which we named C. a. macrodon and C. a. cay. C. a. macrodon has a western and southern Amazon distribution, while C. a. cay has a more southern distribution outside the Amazon basin. (2) In the upper Amazon basin, there is a unique lineage (C. a. macrocephalus) with 1 widely distributed haplotype. The 4 morphological subspecies (C. a. maranonis, C. a. macrocephalus, C. a. peruanus, C. a. pallidus), and maybe a fifth unknown subspecies, described in this area were molecularly undifferentiated at least for the mitochondrial gene analyzed. (3) Our molecular analysis determined that 1 individual of C. robustus fell into the lineage of C. a. macrocephalus. Therefore, this form does not receive any specific name. (4) The animals classified a priori as C. nigritus and C. xanthosternos (because of their morphological phenotypes and by their geographical origins) were clearly differentiated from the other specimens analyzed with the molecular marker employed. Therefore, we consider that these 2 lineages could be assigned the status of full species following the biological species definition. (5) In 2001, Groves described 4 tufted capuchin species (C. apella, C. libidinosus, C. nigritus and C. xanthosternos), while Silva Jr. determined 7 species (C. apella, C. macrocephalus, C. libidinosus, C. cay, C. nigritus, C. robustus and C. xanthosternos). The tests of Swofford-Olsen-Waddell-Hillis, of Shimodaira and Hasegawa and of Templeton did not fit with either of these two classificatory schemes, although Groves' scheme was better with regard to our data than that of Silva Jr. (6) All the temporal splits among the tufted capuchin taxa studied were estimated to have occurred during the last phase of the Pleistocene by using the ρ statistic applied to the median joining haplotype network.

Molecular systematics and phylogeography of the genus Lagothrix (Atelidae, Primates) by means of the mitochondrial COII gene.

We propose the first molecular systematic hypothesis on the origin and evolution of Lagothrix taxa based on an analysis of 720 base pairs of the cytochrome c oxidase subunit II mitochondrial gene in 97 Lagothrix specimens. All the current Lagothrix forms probably descended from the ancestor L. poeppigii or perhaps (less probably) that of L. lugens. We detected at least 2 lineages in L. poeppigii. L. cana and L. lagotricha were determined to be monophyletic and had lower gene diversity levels compared to L. poeppigii and L. lugens. The most basal ancestors of the current L. poeppigii lineages diverged from the other Lagothrix taxa around 2.5 million years ago, at the end of the Pliocene or at the beginning of the Pleistocene. Clearly, L. cana and L. lagotricha were the 2 most recently derived Lagothrix taxa. The diversification within L. lugens and L. poeppigii may coincide with the first and second Pleistocene glacial periods, respectively, while the diversification within L. cana and L. lagotricha could have occurred in the last 400,000 years, coinciding with the climatological changes provoked by the Illinois-Riss (third) and Wisconsin-Würm (fourth) glaciations.