Romain Nattier
email: nattier@mnhn.fr

Claire Capdevielle-Dulac

Catherine Cassildé

Arnaud Couloux

Corinne Cruaud

Gilbert Lachaume

Gerardo Lamas

Jean-François Silvain

Phylogeny and diversification of the cloud forest Morpho sulkowskyi group (Lepidoptera, Nymphalidae) in the evolving Andes ROMAIN NATTIER

The monophyletic Morpho sulkowskyi butterfly group, endemic of Andean cloud forests, was studied to test the respective contributions of Mio-Pliocene intense uplift period and Pleistocene glacial cycles on Andean biodiversity. We sampled nine taxa covering the whole geographical range of the group. Two mitochondrial and two nuclear genes were analysed using a Bayesian method. We established a dated phylogeny of the group using a relaxed clock method and a wide-outgroup approach. To discriminate between two hypotheses, we used a biogeographical probabilistic method. Results suggest that the ancestor of the Morpho sulkowskyi group originated during the Middle-Late Miocene uplift of the Eastern Cordillera in northern Peru, consistent with the divergence time of the Chusquea bamboos, the present hostplants of the group. Biogeographical inference suggests that the M. sulkowskyi and M. lympharis clades diverged in the northern Peruvian Andes. The subsequent divergences, from the Late Miocene to the Late Pliocene, should have resulted from a dispersal towards the Northern Andes (M. sulkowskyi clade), after the closure of the West Andean Portal separating the Central and Northern Andes, and a southwards dispersal along the Peruvian and Bolivian Eastern Cordilleras (M. lympharis clade). Only a few divergences occurred at the very end of the Pliocene or during the Pleistocene, a period when the more recent uplifts interfered with Pleistocene glacial cycles.

INTRODUCTION

The Tropical Andes, the richest and most diverse region on Earth [START_REF] Mittermeier | Hotspots revisited[END_REF]), are a good example of interactions between mountain building and diversification processes that decisively affected present biodiversity patterns [START_REF] Hoorn | Biodiversity from mountain building[END_REF]. To explain the diversification of floras and faunas throughout the vast and complex Andean mountains, different processes were considered, including dispersal and subsequent diversification driven by the rise of mountain ranges that create connections, barriers and new ecological belts, as well as more recent cycles of areas fragmentation and expansion driven by Pleistocene climatic oscillations (e.g., [START_REF] Descimon | Origins of lepidopteran faunas in the high tropical Andes[END_REF][START_REF] Pennington | The role of immigrants in the assembly of the South American rainforest tree flora[END_REF][START_REF] Wahlberg | Colonization of and radiation in South America by butterflies in the subtribe Phyciodina (Lepidoptera: Nymphalidae)[END_REF][START_REF] Antonelli | Tracing the impact of the Andean uplift on Neotropical plant evolution[END_REF][START_REF] Couvreur | Origin and global diversification patterns of tropical rain forests: inferences from a complete genus-level phylogeny of palms[END_REF][START_REF] Condamine | Biogeographic and diversification patterns of Neotropical Troidini butterflies (Papilionidae) support a museum model of diversity dynamics for Amazonia[END_REF][START_REF] Blandin | Evolution and diversification of Neotropical butterflies: Insights from the biogeography and phylogeny of the genus Morpho Fabricius, 1807 (Nymphalidae: Morphinae), with a review of the geodynamics of South America[END_REF]. That recent processes (Pleistocene glacial cycles) played a major role, or not, in the diversification of Andean lineages is a matter of debate [START_REF] Rull | Speciation timing and neotropical biodiversity: the Tertiary-Quaternary debate in the light of molecular phylogenetic evidence[END_REF][START_REF] Rull | Biodiversity, mountains and climate change[END_REF][START_REF] Garzón-Orduña | Timing the diversification of the Amazonian biota: butterfly divergences are consistent with Pleistocene refugia[END_REF], 2015). Various studies, based on robust time-calibrated phylogenies, demonstrated that several plant and animal lineages diversified during the Mio-Pliocene intense uplift period (e.g., [START_REF] Chaves | Diversification in Adelomyia hummingbirds follows Andean uplift[END_REF][START_REF] Fiedler | Past Dynamics of Speciation in Andean Mountain Rainforests[END_REF][START_REF] Castroviejo-Fisher | Neotropical diversification seen through glassfrogs[END_REF][START_REF] Luebert | Effects of mountain formation and uplift on biological diversity[END_REF][START_REF] Sanín | The Neogene rise of the tropical Andes facilitated diversification of wax palms (Ceroxylon: Arecaceae) through geographical colonization and climatic niche separation[END_REF]. However, the role of Pleistocene geoclimatic dynamism has been suggested by other studies (e.g., [START_REF] Garcı Á-Moreno | A Case of Rapid Diversification in the Neotropics: Phylogenetic Relationships among Cranioleuca Spinetails (Aves, Furnariidae)[END_REF][START_REF] Chesser | Evolution in the High Andes: The Phylogenetics of Muscisaxicola Ground-Tyrants[END_REF][START_REF] Casner | Patterns and timing of diversification in a tropical montane butterfly genus, Lymanopoda (Nymphalidae, Satyrinae)[END_REF]. As suggested by meta-analyses, the Mio-Pliocene and Pleistocene hypotheses are complementary [START_REF] Turchetto-Zolet | Phylogeographical patterns shed light on evolutionary process in South America[END_REF][START_REF] Rull | Biodiversity, mountains and climate change[END_REF].

Several works on diversification in the Andes focussed on butterflies or moths (e.g., [START_REF] Adams | Speciation in the pronophiline butterflies (Satyridae) of the northern Andes[END_REF][START_REF] Descimon | Origins of lepidopteran faunas in the high tropical Andes[END_REF][START_REF] Willmott | Systematics of Hypanartia (Lepidoptera: Nymphalidae: Nymphalinae), with a test for geographical speciation mechanisms in the Andes[END_REF][START_REF] Hall | Montane speciation patterns in Ithomiola butterflies (Lepidoptera: Riodinidae): are they consistently moving up in the world[END_REF][START_REF] Elias | Out of the Andes: patterns of diversification in clearwing butterflies[END_REF][START_REF] Casner | Patterns and timing of diversification in a tropical montane butterfly genus, Lymanopoda (Nymphalidae, Satyrinae)[END_REF][START_REF] Strutzenberger | Temporal patterns of diversification in Andean Eois, a species-rich clade of moths (Lepidoptera, Geometridae)[END_REF][START_REF] Matos-Maraví | Systematics and evolutionary history of butterflies in the "Taygetis clade" (Nymphalidae: Satyrinae: Euptychiina): Towards a better understanding of Neotropical biogeography[END_REF][START_REF] De-Silva | Diversification of clearwing butterflies with the rise of the Andes[END_REF]. The diversification of the Neotropics-endemic genus Morpho Fabricius, 1807 (Nymphalidae), that includes the emblematic, metallic blue butterflies, was studied by [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF], and its relation with the geodynamics of the Andes was discussed by [START_REF] Blandin | Evolution and diversification of Neotropical butterflies: Insights from the biogeography and phylogeny of the genus Morpho Fabricius, 1807 (Nymphalidae: Morphinae), with a review of the geodynamics of South America[END_REF]. This monophyletic genus [START_REF] Cassildé | Phylogeny of the genus Morpho Fabricius, 1807, revisited (Lepidoptera, Nymphalidae)[END_REF][START_REF] Cassildé | Phylogeny of the genus Morpho Fabricius 1807: insights from two mitochondrial genes (Lepidoptera: Nymphalidae)[END_REF][START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] originated at ca. 32 Ma [START_REF] Wahlberg | Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary[END_REF] in the east side of the proto-Andes [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] and [START_REF] Blandin | Evolution and diversification of Neotropical butterflies: Insights from the biogeography and phylogeny of the genus Morpho Fabricius, 1807 (Nymphalidae: Morphinae), with a review of the geodynamics of South America[END_REF]. The genus diversified into species living in lowland forests, low mountain forests, and cloud forests, during a period of major and complex orogenic processes, high elevations being attained at different times in Central Andes and Northern Andes, as well as in western and eastern cordilleras [START_REF] Garzione | Rise of the Andes[END_REF][START_REF] Sempere | New insights into Andean evolution: An introduction to contributions from the 6th ISAG symposium[END_REF][START_REF] Eude | Controls on timing of exhumation and deformation in the northern Peruvian eastern Andean wedge as inferred from low-temperature thermochronology and balanced cross section[END_REF]. In order to discriminate between the Mio-Pliocene and Pleistocene diversification hypotheses, Andean-endemic cloud forest butterflies should provide ideal models, as they diversified within a single ecological belt that could develop only when cordilleras attained suitable elevations. We have selected a group of common butterflies, the Morpho sulkowskyi group, that includes closely related taxa distributed from Colombia to Bolivia at 1.8-3.5 km altitudes [START_REF] Krüger | Die Morphiden Kolumbiens nebst einigen allgemeinen Bemerkungen über Morphiden[END_REF][START_REF] Schultze | Beobachtungen und Betrachtungen über zwei in ihren Existenz gefährdete Morpho-Arten Kolumbiens, Morpho rhodopteron v. nevadensis und M. sulkowskyi[END_REF][START_REF] Salazar | Contribución al conocimiento de los Morphinae colombianos. IV. Estudio de una población de Morpho sulkowskyi Kollar 1850, en el centro de Colombia (Lepidoptera: Nymphalidae: Morphinae)[END_REF][START_REF] Prieto | Estructura poblacional de Morpho sulkowskyi Kollar, 1850 (Lepidoptera: Nymphalidae) en un sector de la cordillera occidental, departamento del Cauca (Colombia)[END_REF]personal observations in Bolivia and Peru -PB).

Here, we established a phylogeny to clarify the systematics of the group, and we used a dated phylogeny with distribution data to localize the origin of the group in time and space, and then to discriminate two biogeographical scenarios: 1) a recent vicariant diversification of a widespread species, as a result of the Pleistocene glacial cycles; 2) an older diversification, resulting from dispersal throughout the Andes driven by geodynamics processes, with two opposite predictions: i) origin in the Northern Andes (Colombia and Ecuador), with subsequent dispersal towards the Central Andes (Peru and Bolivia); or ii) origin in the Central Andes and dispersal towards the Northern Andes.

MATERIAL AND METHODS

Taxonomy

Morpho sulkowskyi Kollar, 1849 was described from Colombia. Several morphologically very similar taxa were subsequently described, which have been considered either as species or subspecies. Le [START_REF] Moult | Les Morpho D'Amérique du Sud et Centrale[END_REF] identified one polytypic and five monotypic species, but [START_REF] Lamas | Morphinae[END_REF] recognized only two polytypic species and [START_REF] Blandin | The genus Morpho, Lepidoptera Nymphalidae. Part 2. The subgenera Iphixibia, Cytheritis, Balachowskyna, and Cypritis[END_REF][START_REF] Blandin | The Systematics of the Genus Morpho, Fabricius, 1807 (Lepidoptera Nymphalidae: Morphinae)[END_REF]

Field studies

Three recently described taxa from northern Peru (calderoni Blandin & Lamas, [2007], nieva Lamas & Blandin, [2007], zachi Schäffler & Frankenbach, 2009) were discovered on different slopes of the Eastern Cordillera in its northern part [START_REF] Blandin | La découverte des Morpho dans le Pérou septentrional, de la fin du XIXe au début du XXIe siècle[END_REF]. We performed field surveys to check for the allopatry or, alternatively, the sympatry of these taxa.

After a first survey which allowed us to locate a key area close to the Carretera Transandina near the border between the Amazonas and San Martín departments, specimens were collected along two parts of the upper Río Nieva, that are separated by a mountain range that this river cut across through a narrow gorge (Fig. 1, N1 & N2). Specimens were also collected in neighbouring localities, notably in Oso Perdido (Amazonas) and El Afluente (San Martín) (Fig. 1, OP; EF). The collected specimens have been deposited either in the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (MUSM, Lima) or the Muséum National d'Histoire Naturelle (MNHN, Paris).

Molecular sampling

Field sampling along Río Nieva (Fig. 1, N1 & N2) provided specimens of calderoni, nieva and zachi (deposited in the MNHN). Specimens as recent as possible of sulkowskyi Kollar, 1849 (Colombia: Central Cordillera, Medellín area, Antioquia department), sirene Niepelt, 1911 (Ecuador: Baños area, Tungurahua province), selenaris Le Moult & Réal, 1962 (Peru: Carpish area, Huánuco department), lympharis Butler, 1873 (Peru: Pillahuata area, Cuzco department), descimokoenigi Blandin, 1993 (Peru: Cordillera Urubamba, Cuzco department), and eros Staudinger, 1892 (Bolivia: Incachaca area, Chapare, Cochabamba department) were selected from MNHN collections. There were no available specimens of stoffeli Le Moult & Réal, 1962 (Junín department, Peru). The taxon achiras Fisher, 2009, from Abiseo National Park (San Martín department, Peru), could not be studied, as it is not represented in either MUSM or MNHN. Thus, we studied nine taxa and 91 specimens (Appendix S2a and Fig. 2), covering the whole geographical range of the group.

Total DNA was extracted from the legs or the abdomens of the specimens, using a Qiagen DNeasy Tissue kit. Two mitochondrial (Cytochrome oxidase 1 and Cytochrome b) and two nuclear (EF1a -translation elongation factor 1 alpha and RPS5 -ribosomal protein S5) DNA fragments were amplified by polymerase chain reaction. For additional information on sequencing protocols, see Appendix S3.

Molecular phylogenetic analysis

The sequences of the two mitochondrial genes were concatenated for each individual, since both are from mitochondrial DNA. To conduct Bayesian analyses, the substitution model of evolution was estimated using jMODELTEST v 2.1.4 [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF], and the Akaike information criterion corrected for small samples (AICc) [START_REF] Akaike | Information theory and an extension of the maximum likelihood principle[END_REF][START_REF] Akaike | New Look at Statistical-Model Identification[END_REF] was used to select the GTR+G model. Analyses were performed with MrBayes 3.2.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF]. Four Markov chains were run simultaneously for 50 million generations, sampling every 1000 generations to ensure independence of samples. The first 10 000 trees generated were discarded as burn-in and determined empirically from the log-likelihood values using TRACER V1.6 [START_REF] Rambaut | Tracer v1.4[END_REF]. The remaining trees were used to construct 50% majority-rule consensus trees. Two independent runs were performed to check whether convergence was reached on the same posterior distribution and whether the final trees converged on the same topology. The statistical confidence of each node was evaluated by posterior probabilities. Morpho anaxibia (Esper, [1801]) (sub-genus Iphixibia Le [START_REF] Moult | Les Morpho D'Amérique du Sud et Centrale[END_REF] was chosen as outgroup, as this species belongs to one of the two major Morpho clades (see [START_REF] Cassildé | Phylogeny of the genus Morpho Fabricius 1807: insights from two mitochondrial genes (Lepidoptera: Nymphalidae)[END_REF][START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF], while the Morpho sulkowskyi group belongs to the other.

Dating analyses

To minimize the effect of increased mutation rates at the intraspecific level in dating methods [START_REF] Ho | Time dependency of molecular rate estimates and systematic overestimation of recent divergence times[END_REF][START_REF] Ho | Calibrating molecular estimates of substitution rates and divergence times in birds[END_REF], we used the Poisson Tree Processes (PTP) model to infer putative species boundaries [START_REF] Zhang | A general species delimitation method with applications to phylogenetic placements[END_REF]. This method models speciation or branching events in terms of the number of substitutions, and can use the phylogenetic tree directly without needing the difficult and error prone procedures of time calibration required by other methods such as GMYC [START_REF] Zhang | A general species delimitation method with applications to phylogenetic placements[END_REF]. Analyses were conducted for CO1 gene using the PTP web server (http://species.h-its.org/ptp/) to select one individual per cluster for dating analyses.

Because no suitable fossils for direct calibrations exist within our study group, and in order to be fully independent from regional geological knowledge, we attempted to use a balanced approach to calibrate our analyses using a wide-outgroup approach, which is considered to be a reasonable alternative [START_REF] Hedges | Precision of molecular time estimates[END_REF][START_REF] Strijk | In and out of Madagascar: Dispersal to Peripheral Islands, Insular Speciation and Diversification of Indian Ocean Daisy Trees (Psiadia, Asteraceae)[END_REF]. We extended a published dated genus-level dataset [START_REF] Wahlberg | Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary[END_REF] with our new sequences and sequences available in GenBank for several other Morpho species. The sampling for the dating analysis contained 31 Nymphalidae butterflies (including 17 Morpho species) and three DNA fragments (CO1, EF1a and RPS5) (Appendix S2b).

To improve the estimates of node ages, we used multiple calibration points [START_REF] Lee | Molecular clock calibrations and metazoan divergence dates[END_REF][START_REF] Wang | Divergence time estimates for the early history of animal phyla and the origin of plants, animals and fungi[END_REF][START_REF] Sauquet | Testing the impact of calibration on molecular divergence times using a fossilrich group: the case of Nothofagus (Fagales)[END_REF] on basal and apical nodes. We also checked whether all calibration points were compatible with the reference topology. The dating analysis was conducted using five secondary calibration points [START_REF] Wahlberg | Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary[END_REF] with the entire dataset (Fig. 3A). The split between Calinaginae (Calinaga davidis Oberthür, 1881) and all other Nymphalidae was set at 75.7 Myr (standard deviation: 7.9 Myr, node A), the split between Euxanthe Hübner, [1819] and Charaxes Ochsenheimer, 1816 at 18.1 Myr (4.6 Myr, node B); the diversification of Satyrini at 52.1 Myr (6.4 Myr, node C); the split between Neope Moore, 1866 and Lethe Hübner, [1819] at 36.6 Myr (5.0 Myr, node D), and the diversification of Morphini + Brassolini at 48.5 Myr (5.0 Myr, node E). The calibration of the node D is consistent with fossil data; two fossil Lethe species are known, the age of the oldest being estimated at ca. 32 Myr [START_REF] Miller | A New Lepidopteran Fossil from the Canyon Ferry Reservoir Deposit in Southwestern Montana (Lepidoptera: Nymphalidae: Satyrinae)[END_REF].

We used a normal distribution for the tree prior to node calibration, which is particularly suitable for modelling secondary calibration points [START_REF] Ho | Calibrating molecular estimates of substitution rates and divergence times in birds[END_REF], with the same standard deviation as the Highest Posterior Density (HPD) of [START_REF] Wahlberg | Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary[END_REF]. Calinaga davidis was set as a sister group of all the other species and calibration nodes were constrained on the topology, while all other relationships were left free to vary, so that topological uncertainty was incorporated into posterior estimates of divergence dates. We used the Bayesian relaxed phylogenetic approach, performed in BEAST 1.7.5 [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF], to estimate the relative age of divergence of the lineages studied. We partitioned the data by gene, with unlinked partitions according to the GTR+G substitution model for each and by codon for the CO1. We used the uncorrelated lognormal relaxed clock model and a Birth Death Process of speciation. We confirmed the results by using two independent analyses over 50 million generations, and sampled every 2500th generation. The two analyses converged on similar posterior estimates. We then used Tracer 1.6 [START_REF] Rambaut | Tracer v1.4[END_REF] to assess convergence, measure the effective sample size of each parameter, and calculate the mean and 95% HPD interval for divergence times. We assessed whether a sample size greater than 200 was achieved for all parameters after the analyses. Results of the two runs were combined with LogCombiner 1.7.5 [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF], and the Maximum Clade Credibility tree was compiled with TreeAnnotator 1.7.5 [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF].

Biogeographical reconstructions

To test the dispersal and subsequent speciation hypotheses, we used a probabilistic method implemented in LAGRANGE [START_REF] Ree | Long-term erosion and exhumation of the "Altiplano Antioqueño", Northern Andes (Colombia) from apatite (U-Th)/He thermochronology[END_REF] using biogeographical speciation scenarios and parameters for dispersal and extinction [START_REF] Ree | A likelihood framework for inferring the evolution of geographic range on phylogenetic trees[END_REF]. Terminal taxa were assigned to one or several of the following geographical areas: COL (Colombian Cordilleras), EEQ (Ecuadorian eastern cordilleras), WNP (Western Cordillera in northern Peru), ENP (Eastern Cordillera in northern Peru), ECP (Eastern Cordillera in central Peru), ESPB (eastern cordilleras in southern Peru and Bolivia). We confined possible dispersals to adjacent areas only, specifically between the following combinations: COL-EEQ, EEQ-WNP, EEQ-ENP, ECP-WNP, ECP-ENP, ECP-ESPB. The BEAST chronogram was used for LAGRANGE calculation and the root of the tree was calibrated at 8.9 Ma, as estimated age of the most recent ancestor of all lineages of the M. sulkowskyi group. Separate analyses were also conducted to determine the ancestral root area, using local optimizations conditional on the root state (considering either single or multiple area ranges, but with only adjacent areas, following [START_REF] Condamine | Diversification patterns and processes of wingless endemic insects in the Mediterranean Basin: historical biogeography of the genus Blaps (Coleoptera: Tenebrionidae)[END_REF]. Three LAGRANGE models were run: an unconstrained model, where dispersals among all areas and any combination of ancestral areas are allowed (M0), and two stepping-stone models where dispersals are prohibited between non-adjacent areas and only combinations of adjacent ancestral areas are allowed: the first with dispersal allowed only from southern to northern adjacent areas (M1), while this constraint does not exist in the second (M2).

RESULTS

Field studies

Sympatry was discovered between nieva and calderoni along the upper valley of the Nieva river (transect N1) and westwards at Oso Perdido (OP), while sympatry between nieva and zachi was observed downstream the Nieva (transect N2), and eastwards near El Afluente (EF) (Fig. 1). Specimens of zachi were never collected or observed with calderoni specimens, and reciprocally. Therefore, it is likely that zachi and calderoni populations are parapatric.

Phylogeny and divergence time estimates

The M. sulkowskyi group forms two clades, one including the taxa sulkowskyi, sirene and nieva (clade sulkowskyi) the other selenaris, lympharis, eros, descimokoenigi, calderoni and zachi (clade lympharis) (Fig. 2). In total, eight clusters were recognized under PTP, corresponding to sulkowskyi, sirene, nieva (two clusters), calderoni + zachi, selenaris, lympharis + descimokoenigi, and eros (Fig. 2). We selected one individual per cluster for dating analyses, except for nieva specimens, which were considered as belonging to a unique cluster. The two combined BEAST runs yielded high effective sample sizes (> 200) for all relevant parameters, indicating adequate sampling of the posterior distribution.

The Morpho sulkowskyi group diverged from the (M. portis (Hübner, [1821]) + M. aega (Hübner, [1822]) + M. aurora Westwood, 1851) clade at 14.2 Ma (95% HPD: 18.1 -10.4) (Fig. 3A). The subsequent divergence into the sulkowskyi clade (S, Fig. 3A) and the lympharis clade (L, Fig. 3A) occurred at 8.9 Ma (95% HPD: 11.85.9 Ma). Within the sulkowskyi clade, two subclades originated at 3.9 Ma (95% HPD: 5.82.1 Ma), one corresponding to the Colombian sulkowskyi, the other to the Ecuadorian sirene and the Peruvian nieva. The divergence of nieva ancestors is estimated at 2.6 Ma (95% HPD: 4.11.2 Ma). Within the lympharis clade, two subclades originated at 3.9 Ma (95% HPD: 5.52.4 Ma): a northern one, corresponding to selenaris and calderoni, and a southern one, including lympharis and eros. It should be noted a topological incongruence concerning the divergence of selenaris, which possibly results from differences in molecular sampling (see Fig. 2 and3); the dating analysis being based on mitochondrial and nuclear markers, its result will be used. The divergence of selenaris and calderoni ancestors is estimated at 3.1 Ma (95% HPD: 4.61.7 Ma). Within the Peruvian-Bolivian subclade, eros ancestors diverged from lympharis ancestors at 2.4 Ma (95% HPD: 3.81.3 Ma).

Biogeographical inference

Adjacent areas optimizations recovered the Eastern Cordillera in northern Peru (ENP) as the most likely root state (logL = -22.11). The M2 stepping stone model had a higher loglikelihood (lnL) than the other models (lnL = -18.04 vs. lnL = -19.75 for M0 and lnL = -22.77 for M1), and only results from this best-fitting model are reported (Fig. 3B).

LAGRANGE inference suggests that the M. sulkowskyi and M. lympharis clades diverged in the northern Peruvian Andes. Later, ancestors of the M. sulkowskyi clade dispersed from northern Peru towards the Ecuadorian Andes, and subsequently towards the Colombian cordilleras (divergence between sirene and sulkowskyi subclades). The more recent divergence of nieva ancestors from the sirene lineage possibly resulted from a dispersal towards the northern end of the Peruvian Eastern Cordillera. Within the M. lympharis clade, the major event was the separation of a northern branch and a southern branch that occurred within the Peruvian Eastern Cordillera. Within the northern branch, calderoni ancestors diverged from selenaris ancestors in the northern end of the Eastern Cordillera, while eros ancestors diverged from the lympharis lineage in Bolivia (Fig. 3B).

DISCUSSION

Taxonomy: towards a clarification

The monophyly of the M. sulkowskyi group was first documented by [START_REF] Cassildé | Phylogeny of the genus Morpho Fabricius 1807: insights from two mitochondrial genes (Lepidoptera: Nymphalidae)[END_REF].

Our results confirm the monophyly and clarify relationships between taxa. Their partition into a sulkowskyi clade and a lympharis clade is congruent with the intuition of Le [START_REF] Moult | Les Morpho D'Amérique du Sud et Centrale[END_REF], who divided the Morpho sulkowskyi group into a sulkowskyi subgroup and a lympharis subgroup. Moreover, our field data show that the sulkowskyi and lympharis clades are represented by sympatric populations in the north of the Peruvian eastern Cordillera (Fig. 1). Therefore, these two clades should be considered as distinct species, according to Lamas (2004) taxonomic choice. However, the analysis from PTP resulted in eight clusters, which might be considered as different species (Fig. 2). Nevertheless, it should be emphasized that the morphologically very similar taxa sulkowskyi and sirene correspond to distinct clusters, while the morphologically different taxa zachi and calderoni are included in a single cluster, as well as are the taxa lympharis and descimokoenigi. On another hand, nieva specimens are distributed between two clusters independently of their spatial origin: in each cluster, there are specimens from N1, N2, and PN collecting areas (Fig. 1 and2). In this context, a final taxonomic decision needs to be supported by several analyses with different genes, combined with morphological data and geographic distribution in an integrative taxonomy approach [START_REF] Dayrat | Towards integrative taxonomy[END_REF][START_REF] Schlick-Steiner | Integrative Taxonomy: A Multisource Approach to Exploring Biodiversity[END_REF]. At the moment, we adopt Lamas's viewpoint, and the studied taxa will be designated as follow: M. sulkowskyi sulkowskyi Kollar, 1849, M. sulkowskyi sirene Niepelt, 1911, M. sulkowskyi nieva Lamas & Blandin, [2007], M. lympharis lympharis Butler, 1873, M. lympharis eros Staudinger, 1892, M. lympharis selenaris Le [START_REF] Moult | Les Morpho D'Amérique du Sud et Centrale[END_REF][START_REF] Moult | Les Morpho D'Amérique du Sud et Centrale[END_REF]M. lympharis calderoni Blandin &Lamas, [2007].

The origin of the group: when and where?

The M. sulkowskyi group originated between 18 and 10 Ma, according to our estimation, or even later, between 10 and 5 Ma, according to [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF]. This incongruence between age estimations may be explained by different methodological choices. Recently, [START_REF] Garzón-Orduña | Incompatible ages for clearwing butterflies based on alternative secondary calibrations[END_REF] emphasized the difficulties of choosing between alternative scenarios based on divergence times estimated by different secondary calibrations that may produce incompatible ages. Moreover, [START_REF] Van Tuinen | Potential for bias and low precision in molecular divergence time estimation of the Canopy of Life: an example from aquatic bird families[END_REF] showed that many methodological factors influence differently the estimation of age for deep nodes and shallow nodes. Therefore, differences in species samples and calibration points may explain why we obtained an older ageca. 48 Ma vs ca. 32 Mathan [START_REF] Wahlberg | Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary[END_REF]. Possibly, we also overestimated other ages, including that of the M. sulkowskyi group and of subsequent divergences. However, we used a wide-outgroup approach and five secondary calibration points selected in [START_REF] Wahlberg | Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary[END_REF], one of them being related to the age of a precisely dated fossil, while [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] calibrated only the root of their tree, using the divergence time of the clade Morpho provided by [START_REF] Wahlberg | Nymphalid butterflies diversify following near demise at the Cretaceous/Tertiary boundary[END_REF]. This method strongly underestimates divergence times, as demonstrated by [START_REF] Sauquet | Testing the impact of calibration on molecular divergence times using a fossilrich group: the case of Nothofagus (Fagales)[END_REF].

Therefore, it is possible that the M. sulkowskyi group diverged earlier than [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] supposed.

From a S-DIVA ancestral area reconstruction, [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] 1880, that occurs in the Sierra Nevada de Santa Marta, northern Colombia), also concluded that the ancestors of all these species originated somewhere along the eastern side of the Andes. All these species occur at elevations lower than 2.0 km, except M. rhodopteron that is found from 0.6 to 2.4 km, and M. sulkowskyi and M. lympharis that live from ca. 1.8 to 3.5 km [START_REF] Blandin | The Systematics of the Genus Morpho, Fabricius, 1807 (Lepidoptera Nymphalidae: Morphinae)[END_REF][START_REF] Blandin | Evolution and diversification of Neotropical butterflies: Insights from the biogeography and phylogeny of the genus Morpho Fabricius, 1807 (Nymphalidae: Morphinae), with a review of the geodynamics of South America[END_REF]. Therefore, we hypothesize that the divergence of the M. sulkowskyi group ancestors resulted from an altitudinal shift and adaptation to the cloud forest belt, in areas where altitudes suitable for the development of cloud forests were attained.

In the southern Central Andes (Boliviasouthern Peru), at least 2.0 km elevations were attained by ca. 20-16 Ma [START_REF] Sébrier | Tectonics and uplift in Central Andes (Peru, Bolivia and northern Chile) from Eocene to present[END_REF][START_REF] Leier | Stable isotope evidence for multiple pulses of rapid surface uplift in the Central Andes, Bolivia[END_REF][START_REF] Saylor | Nonuniform surface uplift of the Andean plateau revealed by deuterium isotopes in Miocene volcanic glass from southern Peru[END_REF], and it has been suggested that cloud forests originated in this area around 20-18 Ma (Sempere et al., 2005). At that time, in northern Peru, the Western Cordillera was lower that southern ranges [START_REF] Picard | Direction and timing of uplift propagation in the Peruvian Andes deduced from molecular phylogenetics of highland biotaxa[END_REF], but it attained locally 3 km elevations around 15 Ma [START_REF] Margirier | Slab flattening, magmatism, and surface uplift in the Cordillera Occidental (northern Peru)[END_REF]. Thus, the divergence of M. sulkowskyi group was possible in either the southern Central Andes or the northern Western Cordillera. However, our biogeographical analysis indicates that the M. sulkowskyi group originated probably in the Eastern Cordillera, in northern Peru. Here, from ca. 24 to 17 Ma, a foredeep depozone existed in the place of the Eastern Cordillera, the uplift of which resulted from an intense horizontal shortening between 17 Ma and 8 Ma [START_REF] Eude | Controls on timing of exhumation and deformation in the northern Peruvian eastern Andean wedge as inferred from low-temperature thermochronology and balanced cross section[END_REF]. Our estimation for the divergence time of the M. sulkowskyi group (ca. 18-10 Ma) is consistent with these data. However, as suitable elevations were attained towards the end of the shortening period, [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] estimation (ca. 10-5 Ma) is also compatible. The main point is that the group should have originated in the northern part of the Peruvian Eastern Cordilleran from low or mid-altitude ancestors, when this mountain range attained cloud forest elevations, around 10 Ma.

Mio-Pliocene or Pleistocene diversification?

The first divergence within the M. sulkowskyi group occurred during the Late Miocene, at ca. 8.9 Ma, probably a very few millions of years after the group originated in the Peruvian Eastern Cordillera. The ancestors of the sulkowskyi clade possibly dispersed towards the Western Cordillera, where cloud forests today exist in large areas near the Ecuadorian border, and southwards as restricted, isolated patches surrounded by cultivated areas (Young & León, 2001). This hypothesis is supported by the discovery in one of these relictual cloud forests (Cutervo area, Naranja, 2300 m; Cajamarca department), in 1998, of four males similar to Colombian and Ecuadorian specimens and consequently identified as sulkowskyi by [START_REF] Blandin | The Systematics of the Genus Morpho, Fabricius, 1807 (Lepidoptera Nymphalidae: Morphinae)[END_REF], who considered sirene as a junior synonym of sulkowskyi (these specimens are housed in MUSM; see Appendix 1, Fig. A). Such a distribution, covering the north of the Peruvian Western Cordillera and the eastern Ecuadorian cordilleras, is not surprising: it is known in other cloud forest groups, for example in the hummingbird genus Metallura Gould, 1847, which has an Andean distribution almost similar to the distribution of the M. sulkowskyi group [START_REF] Benham | Biogeography of the Andean metaltail hummingbirds: contrasting evolutionary histories of tree line and habitat-generalist clades[END_REF].

The divergence of sulkowskyi ancestors from sirene ancestors occurred during the Pliocene, at ca. 3.9 Myr. Their common ancestors should have dispersed from the Peruvian Western Cordillera towards the Ecuadorian eastern cordilleras. Until the Middle Miocene, a lowland corridor situated approximately between 3°S and 5°S, the "Western Andean Portal" (WAP) separated the Ecuadorian Andes from the Peruvian Andes [START_REF] Antonelli | Tracing the impact of the Andean uplift on Neotropical plant evolution[END_REF]Hoorn & Wesselingh, 2010). The WAP landscapes included wetlands, and temporary marine incursions from the Pacific Ocean might have occurred [START_REF] Hungerbühler | Neogene stratigraphy and Andean geodynamics of southern Ecuador[END_REF]. Thus, the WAP constituted a long-lasting barrier for mountain organisms [START_REF] Antonelli | Tracing the impact of the Andean uplift on Neotropical plant evolution[END_REF]. Starting from 1311 Ma, the uplift of mountain ranges closed the WAP [START_REF] Coltorti | Geomorphic and tectonic evolution of the Ecuadorian Andes[END_REF][START_REF] Hungerbühler | Neogene stratigraphy and Andean geodynamics of southern Ecuador[END_REF]. Migration of cloud forest species towards Ecuadorian and Colombian Andes became possible when suitable elevations were attained. [START_REF] Luebert | Phylogenetic insights into Andean plant diversification[END_REF] quote several examples of plants that dispersed from the Central to the Northern Andes, and vice versa, during the Late Miocene and Early Pliocene, following the closure of the WAP (103.8 Ma). For example, the ancestors of species of the genus Puya (Bromeliaceae) that diversified in the high Northern Andes originated in the Central Andes and crossed the WAP around 64 Ma [START_REF] Givnish | Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: Insights from an eight-locus plastid phylogeny[END_REF][START_REF] Givnish | Adaptive radiation, correlated and contingent evolution, and net species diversification in Bromeliaceae[END_REF][START_REF] Jabaily | Historical biogeography and life-history evolution of Andean Puya (Bromeliaceae)[END_REF].

We hypothesize that the ancestors of sirene and sulkowskyi lineages also crossed the WAP during this period, corresponding approximately to the Early Pliocene.

In Colombia, the Western and Central Cordilleras already had rather high elevations at the Oligocene-Miocene boundary (2421 Ma) [START_REF] Duque-Caro | Major structural elements and evolution of northwestern Colombia. Geological and geophysical investigations of continental margins[END_REF][START_REF] Gómez | Development of the Colombian forelandbasin system as a consequence of diachronous exhumation of the northern Andes[END_REF][START_REF] Roddaz | Cenozoic sedimentary evolution of the Amazonian foreland basin system[END_REF]. A major phase of uplift followed, and the rise of the Eastern Cordillera began at that time (Restrepo-Moreno et al., 2009). This cordillera reached 2.02.5 km elevations by 3.7 Ma at the latitude of Bogotá [START_REF] Van Der Hammen | Historia y paleoecología de los bosques montanos andinos neotropicales[END_REF]. As we estimated the divergence of sulkowskyi ancestors from sirene ancestors at ca. 3.9 Myr, a synchronous dispersal from the eastern Ecuadorian slopes towards the three cordilleras is plausible during the Late Pliocene.

The diversification of the lympharis clade occurred during a period encompassing the Pliocene and Pleistocene, so it is difficult to differentiate the roles, on one hand of latitudinal range expansion across existing topographical barriers and subsequent allopatric divergence, on the other of the fragmentation of already established areas provoked by glacial cycles. Our results suggest a north-to-south dispersal of the lympharis lineage after its divergence, in the Peruvian Eastern Cordillera, at ca. 3.9 Ma, followed around 2.4 Ma by the divergence of the southernmost eros lineage, perhaps as a consequence of Late Pliocene or early Pleistocene climatic changes. In our present state of knowledge, we suppose that descimokoenigi populations, which are geographically separated from lympharis populations (Appendix S1), diverged during the Pleistocene.

Within the northern subclade, the divergence of calderoni ancestors also occurred during the Late Pliocene (ca. 3.1 Ma). It possibly resulted from a dispersal towards the north-eastern extremity of the Eastern Cordillera, but the role of climatic changes at the very end of the Pliocene or the beginning of the Pleistocene cannot be excluded. Our field studies provided evidence that zachi and calderoni populations are parapatric in the north-eastern extremity of the Eastern Cordillera. We hypothesize that their divergence resulted from Pleistocene fragmentation. However, it should be emphasized the fact that the easternmost mountain ranges raised during the very last few millions of years, and that they probably attained cloud forest altitudes during the Pleistocene; therefore, dispersal of cloud forest species from older ranges may have been controlled by climate-driven altitudinal shifts of ecosystems.

The divergence of nieva ancestors from the sirene lineage occurred approximately at the Pliocene-Pleistocene transition (ca. 2.6 Ma), logically in the north of the Peruvian Western Cordillera. As nieva is known only from the northernmost part of the Eastern Cordillera, which is separated from the Western Cordillera by the dry and deep Marañón valley, we hypothesize that changes in altitudinal distribution of ecological belts associated to Pleistocene climatic variations after 2.6 Ma may have favoured dispersion between the Western and the Eastern Cordilleras.

On the whole, the expansion and diversification of the M. sulkowskyi group essentially occurred before the Quaternary. However, climate-driven cycles of fragmentation and expansion possibly played a role in the ultimate diversification of the group. An almost similar pattern was observed in the butterfly genus Lymanopoda Westwood, 1851 (Nymphalidae, Satyrinae), a species-rich montane group distributed along the Andes. It includes a majority of cloud forest species, the larvae of which eat montane bamboos, primarily Chusquea species [START_REF] Casner | Patterns and timing of diversification in a tropical montane butterfly genus, Lymanopoda (Nymphalidae, Satyrinae)[END_REF]. The genus first diversified between 15 and 10 Ma, the formation of major clades occurred by 10-8 Ma, and most of the species-level diversification occurred since the end of the Miocene, ca. 6 Ma, more than half of the studied species being of Pleistocene or even post-Pleistocene origin. The diversification of another high elevation (1.94.8 km) Andean species, the hummingbird genus Metallura followed an almost similar pattern, with Mio-Pliocene first divergences, but with the majority (81%) of divergence events occurring during the mid-late Pleistocene [START_REF] Benham | Biogeography of the Andean metaltail hummingbirds: contrasting evolutionary histories of tree line and habitat-generalist clades[END_REF]. Thus, such examples suggest that the precise timing of diversification could differ between Andean groups, Pleistocene climatic cycles having a more or less important influence.

The origin of the group: a relationship with hostplant evolution? [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] supposed that caterpillars of all species of the clade including the M. sulkowskyi group and the (M. portis + M. aega + M. aurora) branch feed on monocot hostplants. The hypothesis of a "monocot clade" is supported by available data for M. portis and M. aega, that feed on Guaduinae and Chusqueinae woody bamboos (Poaceae) [START_REF] Beccaloni | Catalogue of the Hostplants of the Neotropical Butterflies. The Natural History Museum[END_REF], and M. sulkowskyi that feeds on a Chusquea species [START_REF] Heredia | Biología y conservación de Morpho sulkowskyi en Colombia (Lepidoptera: Nymphalidae: Morphinae)[END_REF].

Four other species belong to the clade: M. rhodopteron, M. aurora, M. absoloni, and M. zephyritis [START_REF] Cassildé | Phylogeny of the genus Morpho Fabricius 1807: insights from two mitochondrial genes (Lepidoptera: Nymphalidae)[END_REF]. The former also feeds on Chusquea (Montero [START_REF] Abril | Descripción de los estados immaduros de Morpho rhodopteron nevadensis (Lepidoptera: Nymphalidae: Morphinae)[END_REF]. There is no published information on hostplants for the three other species; however, they are always found associated to dense stands of bamboo (G. Lamas pers. obs.), which makes it reasonable to assume their larvae feed on them. Available data indicate that all other Morpho species feed on eudicot hostplants, while other Morphinae (Antirrheina and Brassolini) feed on monocots [START_REF] Beccaloni | Catalogue of the Hostplants of the Neotropical Butterflies. The Natural History Museum[END_REF]. Consequently, [START_REF] Penz | Diversification of Morpho butterflies (Lepidoptera, Nymphalidae): a re-evaluation of morphological characters and new insight from DNA sequence data[END_REF] suggested that the Morpho clade divergence was associated to a shift to eudicotyledonous larval hostplants, and that the divergence of the monocot clade was associated to a reversal to monocot hostplants. This reversal occurred during the 2515 Ma period (Fig. 3B), when Neotropical woody bamboos probably originated and began to diversify: the ancestors of Chusqueinae and Guaduinae originated during the Early-to-Middle Miocene (ca. 2111 Ma), probably in the Andes [START_REF] Fisher | Phylogenetic studies of plastid evolution in the neotropical woody bamboo genus Chusquea (Poaceae: Bambusoideae: Bambusae)[END_REF][START_REF] Ruiz-Sanchez | Biogeography and divergence time estimates of woody bamboos: insights in the evolution of Neotropical bamboos[END_REF]. [START_REF] Heredia | Biología y conservación de Morpho sulkowskyi en Colombia (Lepidoptera: Nymphalidae: Morphinae)[END_REF] observed that the Colombian M. sulkowskyi feeds, at 2.42.5 km a.s.l., on a species closely related to Chusquea scandens Kunth, 1822 one of the five Chusquea species distributed from Bolivia to Colombia [START_REF] Ohrnberger | The Bamboos of the World. Annotated Nomenclature and Literature of the Species and the Higher and Lower Taxa[END_REF]. The clade including C. scandens diverged between ca 14.5 and 6.5 Myr [START_REF] Fisher | Phylogenetic studies of plastid evolution in the neotropical woody bamboo genus Chusquea (Poaceae: Bambusoideae: Bambusae)[END_REF]; our estimation of the age of the M. sulkowskyi group is consistent with this information.

CONCLUSIONS

Simple relationships between Andean uplift and the diversification of various plant and animal groups, implying pre-Pleistocene driving processes, have been supposed by various authors. [START_REF] Doan | A south-to-north biogeographic hypothesis for Andean speciation: evidence from the lizard genus Proctoporus (Reptilia, Gymnophthalmidae)[END_REF], for example, proposed the South-to-North Speciation Hypothesis, where the process of speciation should be related to the south-to-north progression of uplift throughout the Andes. Other authors emphasized the possible role of a rapid uplift that occurred during the Late Miocene and Early Pliocene, but often without establishing clear links between dated divergences and local geologic events (e.g., [START_REF] Casner | Patterns and timing of diversification in a tropical montane butterfly genus, Lymanopoda (Nymphalidae, Satyrinae)[END_REF][START_REF] Mulch | Late Miocene climate variability and surface elevation in the central Andes[END_REF][START_REF] Matos-Maraví | Systematics and evolutionary history of butterflies in the "Taygetis clade" (Nymphalidae: Satyrinae: Euptychiina): Towards a better understanding of Neotropical biogeography[END_REF][START_REF] Lagomarsino | The abiotic and biotic drivers of rapid diversification in Andean bellflowers (Campanulaceae)[END_REF]. From a geological point of view, the concept of a progressive, general south-to-north uplift is an oversimplified view of a much more complex reality [START_REF] Sempere | New insights into Andean evolution: An introduction to contributions from the 6th ISAG symposium[END_REF]. In the Central Andes, palaeoelevation histories differ not only between the south and the north, but also between the Western and the Eastern Cordilleras, notably in northern Peru [START_REF] Picard | Direction and timing of uplift propagation in the Peruvian Andes deduced from molecular phylogenetics of highland biotaxa[END_REF][START_REF] Eude | Controls on timing of exhumation and deformation in the northern Peruvian eastern Andean wedge as inferred from low-temperature thermochronology and balanced cross section[END_REF][START_REF] Margirier | Slab flattening, magmatism, and surface uplift in the Cordillera Occidental (northern Peru)[END_REF]. The idea that the Northern Andes, as a whole, uplifted later than the Central Andes, as suggested by [START_REF] Doan | A south-to-north biogeographic hypothesis for Andean speciation: evidence from the lizard genus Proctoporus (Reptilia, Gymnophthalmidae)[END_REF], and often admitted by other authors, is not supported by geological studies, that also demonstrate that the timing of palaeo-elevation differed between the three Colombian Cordilleras (Restrepo-Moreno et al., 2009). Consistent

  only one (Appendix S1). In the present state of knowledge, we recognize 11 taxa of the species-group: sulkowskyi Kollar, 1849 (Colombian cordilleras); lympharis Butler, 1873 (from southern Peru (Cuzco department) to Bolivia (La Paz department)); eros Staudinger, 1892 (Bolivia, Cochabamba department); sirene Niepelt, 1911 (eastern Ecuador); selenaris Le Moult & Réal, 1962 (northern Peru, upper Huallaga valley, Huánuco department); stoffeli Le Moult & Réal, 1962 (central Peru, Pasco and Junín departments); descimokoenigi Blandin, 1993 (southern Peru, Cuzco department); nieva Lamas & Blandin, [2007] (northern Peru, Amazonas and San Martín departments); calderoni Blandin & Lamas, [2007] (Amazonas department); achiras Fisher, 2009 (northern Peru, Abiseo National Park, San Martín department); zachi Schäffler & Frankenbach, 2009 (northern Peru, San Martín department). Diagnostic characters are indicated and taxa are figured, with their geographical distribution, in Appendix S1.

  concluded that M. sulkowskyi and its relatives M. aurora, M. aega and M. portis originated in what they called the Eastern Andean region, an immense area extending from Colombia to southern Bolivia. Blandin & Purser (2013), taking into account the other M. sulkowskyi and M. lympharis relatives (M. absoloni May, 1924, that lives in Peruvian southern and central lowlands and Andean piedmonts, M. zephyritis, Butler, 1873, that lives along the Andes in southern Peru and Bolivia, from lowlands to low Andean slopes, and M. rhodopteron, Godman & Salvin,

  with many other examples, notably the clearwing Oleriina butterflies[START_REF] De-Silva | Diversification of clearwing butterflies with the rise of the Andes[END_REF], the M. sulkowskyi group illustrates the diversity of diversification histories throughout the Andes. It also demonstrates that Mio-Pliocene orogenic and Pleistocene climatic diversification drivers should not be opposed.

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Topology obtained by Bayesian inference and clusters recognized after analyses conducted for Cytb on the PTP web server (http://species.h-its.org/ptp).

Acknowledgements

This work was conducted within the framework of a cooperation agreement between the Muséum National d'Histoire Naturelle (Paris) and the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos (Lima). It was part of the project "Géographie de la Biodiversité des Lépidoptères dans le nord du Pérou", which received financial support from the Muséum National d'Histoire Naturelle (Programme Pluri-Formations "Etat et structure phylogénétique de la biodiversité actuelle et fossile", 20052009). This work is part of the project @ SPEED-ID "Accurate SPEciEs Delimitation and IDentification of eukaryotic biodiversity using DNA markers" proposed by F-BoL, the French Barcode of Life initiative. This work was made possible thanks to research permits granted by the Dirección de Gestión Forestal y de Fauna Silvestre and the Dirección General Forestal y de Fauna Silvestre, Ministerio de Agricultura y Riego, Peru (autorizaciones: 012-2008-INRENA-IFFS-DCB; 08-2009-AG-DGFFS; 0089-2010-AG-DGFFS-DGEFFS; 0201-2011-AG-DGFFS-DGEFFS; 0201-2013-MINAGRI-DGFFS/DGEFFS). We thank Amanda Fisher for providing her thesis chapter on Chusquea evolution, and for interesting discussions, Jean-François Le Crom, and Stéphane Attal for information on localities in Colombia and Ecuador. At different steps of the drafting process, Marianne Elias, Nicolas Chazot, Mathieu Chouteau, and Fabien Condamine provided useful comments and methodological suggestions. Mathieu Chouteau also contributed to the preparation of the map in Fig. 1. We also warmly thank Thierry

Sempere and Martin Roddaz for stimulating discussions on the highly complex history of the Andes, for providing specialized geological literature, and for reviewing a first draft of the manuscript. We also thank Helen McCombie for improving the English.

Supporting Information

Additional Supporting Information may be found in the online version of this article: Appendix S1. The Morpho sulkowskyi group: geographical distribution and taxonomy.

Appendix S2.

List of all taxa included in the analyses and their Genbank accession numbers: a -phylogenetic analysis and species delimitation; bdating analysis. In GenBank, geographical origins of many specimens are not available (n/a).

Appendix S3. Additional information on sequencing protocols.


Daniel Scott-Algara

Josiane Warszawski

Jérôme Le Chenadec

Céline Didier

Thomas Montange

Jean-Paul Viard

Catherine Dollfus

Véronique Avettand-Fenoel

Christine Rouzioux

Stéphane Blanche

Florence Buseyne
email: florence.buseyne@pasteur.fr

ethnicityin perinatally HIV-1-infected youths -The ANRS-EP38-IMMIP Study

Keywords: perinatal HIV infection, youths, T-cell proliferation, dendritic cells, sCD14, CD4 T cells p

p. 4

Introduction

HIV-specific T cells are important correlates of viral control and delayed disease progression in untreated individuals and may play an important role in future strategies targeting the viral reservoir [START_REF] Deeks | Towards an HIV cure: a global scientific strategy[END_REF] . Understanding their restoration and maintenance is important, especially in patients with a perinatally-acquired infection whose immune system developed in the presence of active HIV-1 replication. We previously reported that Gag-specific CD4 and CD8 T-cell proliferation was more frequently detected in youths of black ethnicity than in those of other ethnicities 2 . These differences in T-cell responses were observed for youths born in the same country, France, with similar routes of HIV infection,similaraccess to health care and whoseimmune systemsweresubjected to asimilar environmental infectious pressure during their entire life. These patients offer a unique opportunity to characterize ethnicityrelated differences in immune restoration, without the confounding effects usually associated with the study of people of diverse geographic origins.

We observed that the levels of myeloid cells and related plasma markers differed between black patients and patients from other ethnic groups.Dendritic cells affect T-cell responses through antigen presentation, secretion of pro-inflammatory molecules, and transmission of HIV particles to susceptible target cells [START_REF] Manches | Dendritic cells in progression and pathology of HIV infection[END_REF] . Thus, we aimed here to study the association between dendritic cellsandGag-specific T-cell proliferation, focusing on patients with undetectable plasma HIV RNA at the time of the study.

p. 5

Materials and methods

Patients

The ANRS-EP38-IMMIP study included 93 patients who acquired HIV-1 infection during the perinatal period and were at least 15 years old at the time of the study [START_REF] Avettand-Fenoel | Relationships between HIV disease history and blood HIV-1 DNA load in perinatally infected adolescents and young adults: the ANRS-EP38-IMMIP Study[END_REF][START_REF] Blanche | Naive T lymphocytes and recent thymic emigrants are associated with HIV-1 disease history in French adolescents and young adults infected in the perinatal period: The ANRS-EP38-IMMIP study[END_REF] . The study was approved by the local ethics committees. All patients, and their legal guardians for those under 18 years of age, received written information and signed an informed consent form.

This study was approved by the "Comité de protection des personnes Ile-de-France II" (registration number 06-09-08), authorized by the "Direction Générale de la Santé" (authorization number 2006-AOO142-49). The ANRS-EP38-IMMIP study is registered as an observational study at www.clinicaltrials.gov under identifier NCT01055873. A single blood sample was drawn for virological and immunological assays. At the time of the study, the threshold values used in HIV RNAassays depended on the volume of plasma available. The highestthreshold used was 80 copies/mL, and this value was selected asthe cutoff for current HIV RNA detection, as described [START_REF] Avettand-Fenoel | Relationships between HIV disease history and blood HIV-1 DNA load in perinatally infected adolescents and young adults: the ANRS-EP38-IMMIP Study[END_REF] . The cutoff of 500 copies/mL was chosen for assessments of virological history, because of changes in HIV RNAquantification assays over time.

Biological analyses

Freshly isolated peripheral blood mononuclear cells (PBMCs) were labeled with 10 µM CFSE (5-6-carboxyfluorescein diacetate succimidyl ester) and stimulated with pools of Gag peptides (NIH #8117). Six days later, the cells were labeled with a combination of anti-CD3-ECD, anti-CD4-PC7, and anti-CD8β-PC5 antibodies (Beckman Coulter, Villepinte, France), and analyzed on a FC500 flow cytometer (Beckman Coulter). CFSE low T cells were quantified for CD4 and CD8 T-cell subpopulations (see [START_REF] Chenadec | Gag-specific CD4 and CD8 T-cell proliferation in adolescents and young adults with perinatally acquired HIV-1 infection is associated with ethnicity -The ANRS-EP38-IMMIP study[END_REF] for gating strategy). Assays were scored positive if the difference in the percentages of CFSE low cells between antigenstimulated and mock-stimulated cells was above 0.8% and the ratio of these percentages was above 4. This dual criterion cutoff was based on assays performed with uninfected donors, as described [START_REF] Chenadec | Gag-specific CD4 and CD8 T-cell proliferation in adolescents and young adults with perinatally acquired HIV-1 infection is associated with ethnicity -The ANRS-EP38-IMMIP study[END_REF] . Patients with positive results in the Gag-specific CD4 T-cell assay are referred to p. 6 as CD4 responders (CD4Rs) and those with negative results for this assay are referred to as CD4 nonresponders (CD4NRs). Similarly, CD8Rs and CD8NRs are defined on the basis of the results of the Gag-specific CD8 T-cell proliferation assay.CD4 T-cell percentages and CD4 T-cell and monocyte counts were determined at the clinical sites as part of the standard follow-up procedure. Myeloid and plasmacytoid dendritic cells (mDCs and pDCs) were studied with a combination of labeled antibodies:FITC-anti-CD14, FITC-anti-CD16, PE-anti-CD85k, PC5-anti-CD33, PC7-anti-CD123(Fig. 1) [START_REF] Blank | Circulating dendritic cell number and intracellular TNF-alpha production in women with type 2 diabetes[END_REF][START_REF] Orsini | Enumeration of human peripheral blood dendritic cells throughout the life[END_REF] .Results were available for 42 patients.Patients with and without dendritic cell data were similar regarding age, CD4 T-cell count, cell-associated HIV DNA levels, time spent with an HIV RNA level < 500 copies and time spent on HAART (data not shown). Plasma samples from 52 out of 53 patients were assayedfor cytokines, chemokines and sCD14, according to the manufacturer's instructions for each kit (references andquantification thresholdsare presented in Supplementary Table 1).

Statistical analysis

Associations between ethnicity and immune parameters were assessed using the Fisher's exact test and the Mann-Whitney test for qualitative and quantitative variables, respectively. Patients were classified according to the detection of Gag-specific CD4 and CD8 T-cell proliferation.Logistic regression was used for univariate and multivariate analyses of data. We took ethnic origin into account, by grouping patients originatingfrom sub-Saharan Africa and the Caribbean and comparing them with those ofother geographic origins (mainland France, North Africa and Asia). The patients are referred to as being of black or other ethnicity. Phylogenetic analyses were carried out on the sequences of the Env and RT genes, to determine virus subtype, as described in [START_REF] Chenadec | Gag-specific CD4 and CD8 T-cell proliferation in adolescents and young adults with perinatally acquired HIV-1 infection is associated with ethnicity -The ANRS-EP38-IMMIP study[END_REF] . The analyses were exploratory andwere, therefore, not corrected for multiple testing. For variables associated with Gag-specific T-cell proliferation with a P value ≤0.20 in univariate analysis, we tested the associations with inclusion of demographic and HIV disease history variables previously found to be associated with T-cell proliferation 2 . We did not build a model with all immunological parameters, as p. 7

these were not quantified for all patients, due to a lack of available samples for some of them.

Analyses were conducted usingSAS software (version 9.2). A P value of <0.05 was considered to indicate statistical significance.

p. 8

Results

Patients

The analyses described here were restricted to the 53 patients who were receiving highly active antiretroviral treatment (HAART)and had undetectable plasma HIV RNA at the time of the study, and for whom a valid proliferation assay had been carried out. Twenty-four of the patients were male, and 19 were of black ethnicity. At the time of the study, the median age of the patients was 18 years (interquartile range (IQR): 16-19 years), patients had been on HAART for a median cumulative duration of 9.1 years (IQR: 7.6-9.8 years), had a median CD4 T-cell count of 635 cells/µl (IQR: 522-918 cells/µl) and a median duration of HIV RNA < 500 copies/ml of 3.0 years (IQR: 1.3-6.3 years).

Gag-specific CD4 T-cell proliferationis associated with higher peripheral blood dendritic cell levels

Black patients had higherpercentages and absolute counts of mDCs than patients of other ethnic groups(Table 1). They tended to have higherpercentages of pDCs and lower monocytecounts. We compared the levels of these antigen-presenting cells (APCs) between patients with and without Gag-specific CD4 T-cell proliferation(CD4Rs and CD4NRs, respectively). Data are presented for the two ethnic groups in Fig. 2 and results from the logistic regression are reportedin panels J-K.CD4Rs had significantly higher percentages and absolute counts of mDCs and pDCs in the peripheral blood thanCD4NRs (Fig. 2A-D). The peripheral blood monocyte count was not associated with Gag-specific CD4 T-cell proliferation (Fig. 2E). Gag-specific CD4 T-cell proliferation was associated with the percentage and absolute count of pDCafter adjustment for ethnicity (Fig. 2K). Inclusion of the HIV subtype in the multivariate analysis did not modify the associations (data not shown).

Gag-specific CD4 T-cell proliferation tends to be associated with lower plasma sCD14 levels

p. 9

We then quantified plasma markers related to APC activation (sCD14, neopterin, and CCL2), and type I IFN production (IFN-α and TRAIL) because these moleculesmay suppress or promote T-cell proliferation (Fig. 2F-J). Black patients had significantly lower levels of CCL2 and tended to have lower levels of sCD14, neopterin, and IFN-α than patients of other ethnicities (Table 1). CD4Rs tended to have lower sCD14 levels than CD4NRs. Neither IFNαnor TRAIL plasma levels were associated with Gag-specific T-cell proliferation.

We also assessed the levels of chemokines that direct T-cell and APCmigration to the secondary lymphoid organs in steady-state and inflammatory conditions: CCL3, CCL4, CCL21, CXCL10, and CXCL12 [START_REF] Bromley | Orchestrating the orchestrators: chemokines in control of T cell traffic[END_REF] . Plasma levels of these five chemokines were not related to ethnicity (Table 1). We observed a trend for lower CXCL12 levels in CD4Rs (Fig. 2J). None of the plasma markers were associated with Gag-specific CD4 T cell proliferationafter adjustment for ethnicity (Fig 2K).

Gag-specific CD8 T-cell proliferation tends to beassociated with lower levels of monocyte activation markers in plasma

A similar analysis was conducted for Gag-specific CD8 T-cell proliferation. CD8Rs tended to have higher levels of mDCs than CD8NRs, by univariate analysis only (Fig. 3).

CD8Rs had significantly lower plasma concentrations of sCD14 and CCL2 than CD8NRs (Fig. 3F and3H). CCL2 was notassociated with Gag-specific CD8 T-cell proliferation, after adjustment for ethnicity and duration of HIV RNA < 500 copies/ml, but we observed a trend for lowersCD14(Fig. 3K).

p. 10

Discussion

The ANRS-EP38-IMMIP focused on the first generation of youths with perinatally acquired HIV-1 infection. We previously reported that Gag-specific CD4 and CD8 T-cell proliferationismore frequent in black patients than in patients from other groups [START_REF] Chenadec | Gag-specific CD4 and CD8 T-cell proliferation in adolescents and young adults with perinatally acquired HIV-1 infection is associated with ethnicity -The ANRS-EP38-IMMIP study[END_REF] .We now expand these findings by showing that Gag-specific CD4 T-cell proliferation isassociated with higher levels of pDCs.

The association between pDC levels and HIV-specific CD4 T-cell proliferationmay reflect the in vivo cross-talk between innate and adaptive immunity [START_REF] Swiecki | The multifaceted biology of plasmacytoid dendritic cells[END_REF] . DC depletion in infected children is similar to that of patients infected as adults [START_REF] Azzoni | Dendritic and natural killer cell subsets associated with stable or declining CD4 cell counts in treated HIV-1-infected children[END_REF][START_REF] Zhang | Differential restoration of myeloid and plasmacytoid dendritic cells in HIV-1-infected children after treatment with highly active antiretroviral therapy[END_REF][START_REF] Badolato | Type I interferon-dependent gene MxA in perinatal HIV-infected patients under antiretroviral therapy as marker for therapy failure and blood plasmacytoid dendritic cells depletion[END_REF][START_REF] Usuga | Characterization of quantitative and functional innate immune parameters in HIV-1-infected Colombian children receiving stable highly active antiretroviral therapy[END_REF][START_REF] Selvaraj | Defective dendritic cell response to Toll-like receptor 7/8 agonists in perinatally HIV-infected children[END_REF] , and these cells had reduced T-cell stimulatory activity [START_REF] Chougnet | Influence of human immunodeficiency virus-infected maternal environment on development of infant interleukin-12 production[END_REF][START_REF] Hughes | Dendritic cell anergy results from endotoxemia in severe malnutrition[END_REF] . Upon treatment, viral suppression allows the quantitative and functional restoration of dendritic cells, but to levels usually below those of uninfected controls [START_REF] Chehimi | Baseline viral load and immune activation determine the extent of reconstitution of innate immune effectors in HIV-1-infected subjects undergoing antiretroviral treatment[END_REF][START_REF] Altfeld | DCs and NK cells: critical effectors in the immune response to HIV-1[END_REF] . Our data suggest that functional restoration of CD4 HIV-specific T cells may be limited by that of DCs.

In vivo, non-progressive HIV infection is associated with a highcapacity of dendritic cells to stimulate T cell proliferation [START_REF] Huang | Leukocyte immunoglobulin-like receptors maintain unique antigen-presenting properties of circulating myeloid dendritic cells in HIV-1infected elite controllers[END_REF] .Inaddition, DCs induce a higher frequency of responding T cells than PBMCs loaded with the same antigen in in vitro assays [START_REF] Betts | The functional profile of primary human antiviral CD8+ T cell effector activity is dictated by cognate peptide concentration[END_REF][START_REF] Huang | Multiple T-cell responses to human immunodeficiency virus type 1 are enhanced by dendritic cells[END_REF] , and the transduction of APCs with costimulatory molecules enhances HIV-specific T-cell proliferation [START_REF] Bukczynski | Enhancement of HIV-specific CD8 T cell responses by dual costimulation with CD80 and CD137L[END_REF][START_REF] Keersmaecker | The combination of 4-1BBL and CD40L strongly enhances the capacity of dendritic cells to stimulate HIV-specific T cell responses[END_REF] . In adults, pDCs and HIV-specific CD8 T-cell frequencies are independent predictors of the viral set-point in the context of planned ART interruption, and the effect of pDCs appears to be related to the presence of HIV-specific CD8 T cells [START_REF] Papasavvas | Plasmacytoid dendritic cell and functional HIV Gag p55-specific T cells before treatment interruption can inform set-point plasma HIV viral load after treatment interruption in chronically suppressed HIV-1+ patients[END_REF] . However, in our study we observed the association between pDCs and HIV-specific CD4 T-cell proliferation but not between pDCs and HIV-specific CD8 T-cell proliferation.

We report the association between Gag-specificCD4 and CD8 T-cell proliferation and low levels of myeloid cell activation plasma markers. sCD14 exerts a immunoregulatory function:itsuppressed mitogen and antigen-specific T-cell proliferation in PBMCs upon binding to the surface of monocytes [START_REF] Lue | Engagement of CD14 on human monocytes terminates T-cell proliferation by delivering a negative signal to T cells[END_REF][START_REF] Rey-Nores | Soluble CD14 acts as a negative regulator of human T cell activation and function[END_REF] . Despite previous reports showing alterations in Tp. 11 cell migration in HIV-infected patients [START_REF] Combadière | The chemokine receptor CX3CR1 controls homing and anti-viral potencies of CD8 effector-memory T lymphocytes in HIV-infected patients[END_REF][START_REF] Brainard | Decreased CXCR3+ CD8 T cells in advanced human immunodeficiency virus infection suggest that a homing defect contributes to cytotoxic T-lymphocyte dysfunction[END_REF][START_REF] Meditz | SDF-1 alpha is a potent inducer of HIV-1-specific CD8+ T-cell chemotaxis, but migration of CD8+ T cells is impaired at high viral loads[END_REF][START_REF] Perez-Patrigeon | HIV infection impairs CCR7dependent T-cell chemotaxis independent of CCR7 expression[END_REF] , we observed only a modest trend for the association between Gag-specific T cell proliferation and plasma chemokine levels. This may be because T lymphocytes interact with chemokines bound to extracellular matrix molecules or to the surface of endothelial and stromal cells [START_REF] Bromley | Orchestrating the orchestrators: chemokines in control of T cell traffic[END_REF] orour study lacked sufficient statistical powerto detect modest associations.

Finally, we report that youths of black ethnicity have higher levels of dendritic cells and lower levels of activation and inflammation markers than youths of other ethnicities.A study that compared adults from Uganda and the UK found no difference in dendritic cell levels [START_REF] Jones | Comparative loss and maturation of peripheral blood dendritic cell subpopulations in African and non-African HIV-1-infected patients[END_REF] . However, lower CCL2 levels were reported in African American than in Caucasian patients [START_REF] Shebl | Increased levels of circulating cytokines with HIV-related immunosuppression[END_REF] . Both DCs and immune activation levels may contribute to stronger T-cell responses in patients of black ethnicity in our study. This may also be truefor other studies that reported stronger HIV orHCV-specific T-cell responses in black individuals than in individuals from other races living in a similar environment [START_REF] Sugimoto | Influence of ethnicity in the outcome of hepatitis C virus infection and cellular immune response[END_REF][START_REF] Sharp | Higher frequency of HIV-1-specific T cell immune responses in African American children vertically infected with HIV-1[END_REF][START_REF] Golden-Mason | Programmed death-1 expression is increased on immunocytes in chronic hepatitis C virus and predicts failure of response to antiviral therapy: Race-dependent differences[END_REF][START_REF] Perez-Losada | Ethnic differences in the adaptation rate of HIV gp120 from a vaccine trial[END_REF] .These differences in plasma CCL2 levels observed between ethnic groups may be related to different frequencies of patients carrying genetic polymorphisms. As an example, the -2581A/G polymorphism in the promoter of theccl2 gene affects protein expression is associated with tuberculosis risk in certain ethnic groups [START_REF] Zhang | The -2518A/G polymorphism in the MCP-1 gene and tuberculosis risk: A meta-analysis[END_REF] . Other polymorphisms in the ccl2gene coding sequence have been described in West-Africans [START_REF] Thye | MCP-1 promoter variant -362C associated with protection from pulmonary tuberculosis in Ghana, West Africa[END_REF] . To the best of our knowledge, the impact of these genetic polymorphisms on the quantification of plasma CCL2 levels by the currently available assays is unknown, and such knowledge would be relevant for further biomarkers studies.Overall, our results suggest that ethnicity may affect the capacity of DCs to produce immune activation molecules and to respond to activation and inflammatory signals.

The strength of our study has been to compare biological differences among patients of various ethnic origins, who shared similar exposure to the virus, access to treatment and environmental factors. This study has some limitations. First, we cannot exclude that some differences in immune restoration may be affected by non-biological factors such as p. 12 socioeconomic factors that affectaccess to care, although there were no major differences in HIV infection history foreitherethnic group, as discussed in 2 . Unfortunately, there were no genetic studies, and no HLA-tissue typing, as specific consent is required for genetic investigations in France. We did not include such tests as a requirement for inclusion in the study, as we were concerned that this would have reduced the willingness of subjects to participate. For the biological variables presented in this study, patients fromsub-Saharan Africa had values similar to those from patients from the Caribbean, but different from those of patients of other ethnic origins (data not shown). Therefore, these patients were compared to all patients of other ethnicities. The small number of patients in the Caribbean, North Africa and Asia groups precluded their independent analysis. Finally, this was an exploratory analysis; the sample size was sufficient for the results to be conclusive for some but not all immune parameters tested.

We provide new information on higher dendritic levels related toboth the black ethnic background andthe recovery of HIV-specific CD4 T-cell proliferation under cART. Our results highlight the need to expand our knowledge on the impact of ethnicity on immunemediated clinical events, such as the immune reconstitution syndrome, and on potentially useful immune markers for clinical management, such as sCD14.

p. 13

Acknowledgments and funding

Peptides were provided by the NIH AIDS Research and Reference Reagent Program.

We thank all the patients who agreed to participate in this study. We would also like to thank the nurses and staff members from the various clinical sites. We thank Sandrine Leveillé (Hôpital Robert Debré), Geneviève Vaudre (Hôpital Trousseau), Sylvie Tassi (Hôpital Jean Verdier), Nora Boudjoudi (Hôpital Port Royal), Marie-Christine Mourey (Hôpital Necker), Thierry Wack (CESP INSERM U1018), Yassine Benmebarek and Naima Bouallag-Bonnet (former members of CESP INSERM U1018). We thank Yves Rivière for his role in this collaborative work. We would also like to thank Elisabeth Monchâtre, Pauline Louche and Céline Clairet for expert technical assistance. This text has been verified by a native English speaker.

This work was supported by the 2006-232 and 2009-165 grants from the "Agence Nationale de recherche sur le SIDA et les Hépatites" (ANRS, www.anrs.fr) and by the "Fondation AREVA" (http://www.areva.com/FR/groupe-910/fondation-implication-societalepour-un-developpement-durable.html). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

Competing interests

The authors have no competing interests to declare.

Footnotes

Additional files

SupplementaryData_ARHR.docx contains Supplementary Table 1.

p. 15