Nicolas Brasero
email: brasero.nicolas@gmail.com

Baptiste Martinet

Denis Michez

Thomas Lecocq

Irena Valterova

Pierre Rasmont

Taxonomic revision of the Sylvarum group of bumblebees using an integrative approach

Keywords: Bumblebees, eco-chemical traits, integrative taxonomy, mitochondrial genetic maker, nuclear genetic maker, Thoracobombus

Recent improvements in taxonomy consider multiple operational criteria. Integrative taxonomy provides a methodological framework merging these multisource approaches.

Bumblebees are considered as a uniform group where their taxonomy remains one of the most difficult. Here, we investigate the taxonomic statuses inside a monophyletic group including six taxa (B. inexspectatus, B. mlokosievitzii, B. ruderarius, B. sylvarum, B. velox and B. veteranus) in the most diverse subgenus of bumblebees: Thoracobombus. We used an integrative approach based on mitochondrial and nuclear genetic markers and eco-chemical traits commonly used in bumblebee taxonomy. For all species, our study shows a clear differentiation in DNA and eco-chemical traits. However, we conserve the subspecies status of B. ruderarius simulatilis and B. sylvarum daghestanicus from the east of Turkey and Iran according to their degree of genetic and eco-chemical differentiation.

Introduction

Species are considered as the fundamental unit in biology and their accurate delimitation is essential respectively in biodiversity classification and in evolutionary and conservation biology [START_REF] Moraes | Importance of taxonomy in biological control[END_REF][START_REF] Paquin | The fundamental importance of taxonomy in conservation biology: the case of the eyeless Cicurina bandida (Araneae : Dictynidae) of central Texas, including new synonyms and the description of the male of the species[END_REF][START_REF] Wheeler | Taxonomy: Impediment or expedient?[END_REF]. Taxonomy inside invertebrate groups is mainly based on morphological approach. However, the common strong homogeneity in morphology may be an obstacle for identification, especially in the case of cryptic species [START_REF] Williams | Unveiling cryptic species of the bumblebee subgenus Bombus s. str. worldwide with COI barcodes (Hymenoptera: Apidae)[END_REF].

Moreover, many species exhibit a high intra-specific variability and cases of convergent color pattern were recorded in sympatry [START_REF] Françoso | An integrative approach identifies a new species of bumblebee (Hymenoptera: Apidae: Bombini) from northeastern Brazil[END_REF]Lecocq et al., 2015a). Therefore, many alternative features and methods such as DNA-based approaches (e.g. pairwise distance thresholds, bootstrap support or reciprocal monophyly), geometric morphometrics of wing shape or semiochemical traits have been used to delineate species [START_REF] Lecocq | Methods for species delimitation in bumblebees (Hymenoptera, Apidae, Bombus): towards an integrative approach[END_REF][START_REF] Martin | Evolution of species-specific cuticular hydrocarbon patterns in Formica ants[END_REF][START_REF] Oleksa | Wing geometric morphometrics and microsatellite analysis provide similar discrimination of honey bee subspecies[END_REF][START_REF] White | Comparison of four species-delimitation methods applied to a DNA barcode data set of insect larvae for use in routine bioassessment[END_REF]. In order to draw a strongly supported taxonomic hypothesis, recent betterment in taxonomy consider multiple operational criteria from the same sampling [START_REF] Gibbs | Integrative taxonomy identifies new (and old) species in the Lasioglossum (Dialictus) tegulare (Robertson) species group (Hymenoptera, Halictidae)[END_REF][START_REF] Lecocq | Molecular and chemical characters to evaluate species status of two cuckoo bumblebees: Bombus barbutellus and Bombus maxillosus (Hymenoptera, Apidae, Bombini)[END_REF][START_REF] Martinet | Following the cold: geographical differentiation between interglacial refugia and speciation in the arcto-alpine species complex Bombus monticola (hymenoptera: Apidae)[END_REF]. The integrative taxonomy based on the Unified Species Concept (USC) provides a methodological framework merging these multisource approaches [START_REF] De Queiroz | Species concepts and species delimitation[END_REF][START_REF] Padial | The integrative future of taxonomy[END_REF][START_REF] Schlick-Steiner | Integrative taxonomy: a multisource approach to exploring biodiversity[END_REF]. The USC argues that all species concepts agree that species exist as separately evolving metapopulation lineages but diverge in their criterion for delimiting species [START_REF] De Queiroz | Species concepts and species delimitation[END_REF]. Therefore, ecological differentiation, phylogenetic divergence, morphological characteristic or reproductive isolation could highlight a metapopulation lineage separation by avoiding false taxonomic conclusions driven by a single trait. Moreover, by considering subspecies as a step in the process of allopatric speciation [START_REF] Mayr | Systematics and the Origin of Species[END_REF], and because of its important value in conservation and evolutionary biology [START_REF] Coppée | Intraspecific variation of the cephalic labial gland secretions in Bombus terrestris (L.) (Hymenoptera: Apidae)[END_REF][START_REF] Phillimore | Are subspecies useful in evolutionary and conservation biology[END_REF][START_REF] Rasmont | An overview of the Bombus terrestris (L. 1758) subspecies (Hymenoptera: Apidae)[END_REF][START_REF] Sackett | Evidence for two subspecies of Gunnison's prairie dogs (Cynomys gunnisoni), and the general importance of the subspecies concept[END_REF][START_REF] Terzo | Intra and interspecific variability of the cephalic labial glands' secretions in male bumblebees: the case of Bombus (Thoracobombus) ruderarius and B. (Thoracobombus) sylvarum Hymenoptera, Apidae[END_REF], subspecies status has been proposed as solution when differentiation is highlighted by a subset of operational criteria. This proposal aims to reduce the underestimate risk of a congruence approach [START_REF] Hawlitschek | Island evolution and systematic revision of comoran snakes: Why and when subspecies still make sense[END_REF][START_REF] Padial | The integrative future of taxonomy[END_REF].

Bumblebees are considered as a complex group where their classification remains one of the most difficult in bees [START_REF] Moure | As Mamangabas Sociais do Brasil (Bombus Latreille) (Hymenoptera, Apoidea)[END_REF][START_REF] Murray | Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs[END_REF]. In this group, integrative taxonomy has considerably improved taxonomic studies and has solved many doubtful cases thanks to operational criteria as DNA (mitochondrial and nuclear) and semio-chemicals [START_REF] Bertsch | Male labial gland secretions and mitochondrial DNA markers support species status of Bombus cryptarum and B. magnus (Hymenoptera, Apidae)[END_REF]Lecocq et al., 2015a;[START_REF] Martinet | Following the cold: geographical differentiation between interglacial refugia and speciation in the arcto-alpine species complex Bombus monticola (hymenoptera: Apidae)[END_REF].

In this paper, we apply an integrative approach to assess the taxonomic status of a group of phylogenetically closely related species in the Thoracobombus Dalla Torre, 1880 subgenus: Radoszkowski, 1877, B. ruderarius (Müller, 1776), B. sylvarum (L., 1761), B. velox [START_REF] Skorikov | Les formes nouvelles des bourdons (Hymenoptera, Bombidae)[END_REF], B. veteranus [START_REF] Fabricius | Entomologica systematica emendata at aucta. Secundum classes, ordines, genera, species adjectis synonimis[END_REF] and Bombus inexspectatus [START_REF] Tkalců | Eine neue Hummel-Art der Gattung Agrobombus Vogt aus dem Alpengebiet[END_REF]. We revise the taxonomy of this group through a decision framework based on differentiation of three operational and independent criteria commonly used in bumblebee taxonomy: a mtDNA marker (COI), a nuDNA marker (PEPCK) and an eco-chemical trait.

B. mlokosievitzii

Materials and methods

Studied taxa

B. ruderarius [START_REF] Müller | Zoologiae Danicae prodromus, seu animalium Daniae et Norvegiae indigenarum characteres, nomina, et synonyma imprimis popularium[END_REF]) is a widespread species distributed from Tunisia in the south to the Arctic Circle in the north (Rasmont et al., 2015a). Four subspecies and one form have been described [START_REF] Rasmont | Catalogue commenté des Bourdons de la région ouest-paléarctique (Hymenoptera, Apoïdea, Apidae)[END_REF]): (i) B. ruderarius ruderarius [START_REF] Müller | Zoologiae Danicae prodromus, seu animalium Daniae et Norvegiae indigenarum characteres, nomina, et synonyma imprimis popularium[END_REF] is characterized by a reddish tail and the rest of the body is black intermixed with more or less greyish hairs (Fig. 1C). They occurred throughout Europe except the Mediterranean region; (ii) B. ruderarius simulatilis Radoszkowski, 1888 with a wide white collare and scutellare bordering a black interalar band, the tergite 1 and 2 completely white and with the tergite 3 black, the tail is red (Fig. 1D). This subspecies occurred in mountainous regions of Anatolia as well as in the Caucasus region in north Iran; (iii) B. ruderarius tunensis [START_REF] Tkalců | Beitrag zur Kenntnis der Hummelfauna der französischen Basses-Alpes (Hymenoptera, Apoidea, Bombinae)[END_REF] is characterized by three yellow bands and a white tail and is endemic to the east of Algeria and north of Tunisia; (iv) B. ruderarius sartus [START_REF] Skorikov | Palaearctic bumble bees. Part I. General biology (including zoogeography) (in russian)[END_REF] with a wide yellowish collare and scutellare bordering a black interalar band, the tergite 1 and 2 completely yellowish and with the tergite 3 black, the tail is red. The eastern limits of the species were known to be the mountains of east Turkestan but other specimens were found in Mongolia [START_REF] Skorikov | Palaearctic bumble bees. Part I. General biology (including zoogeography) (in russian)[END_REF];

(v) B. ruderarius form montanus is characterized by large yellowish bands compared to the dark nominal subspecies (Fig. 1C). They occur from the Cantabrian Mountains to the Pyrenees where they coexist with the nominal subspecies B. ruderarius ruderarius. B. sylvarum (L., 1761) is also a widespread species distributed in the west-palaearctic region but hardly reaches Mongolia (Rasmont et al., 2015a). We considered four subspecies and one form [START_REF] Rasmont | Catalogue commenté des Bourdons de la région ouest-paléarctique (Hymenoptera, Apoïdea, Apidae)[END_REF]): (i) B. sylvarum sylvarum (L., 1761) with three large greyish bands (thorax and abdomen) and thin grey bands (abdomen) and a reddish tail intermixed with grey (Fig. 1F). They occur in the west-Palaearctic region; (ii) B. sylvarum citrinofasciatus [START_REF] Vogt | Studien über das Artproblem. 1. Mitteilung. über das Variieren der Hummeln. I. Teil[END_REF] is characterized by grey yellowish bands compared to the nominal subspecies.

Moreover, the thin grey bands on the abdomen are often absent. They occur in the west of Turkey; (iii) B. sylvarum daghestanicus Radoszkowski, 1877 is characterized by white bands and a reddish tail (Fig. 1E). This subspecies is distributed in the east of Turkey, the Caucasus region as well as in Iran; (iv) B. sylvarum rogenhoferi Dalla Torre, 1882 with white yellowish bands, a red tail and without thin grey bands on the abdomen is restricted to south Italy and Sicily; (v) B. sylvarum form nigrescens Pérez, 1879 is considered as the melanic form inside B. sylvarum species. They are completely black with a reddish tail and with or without intermixed grey hairs on the thorax (Fig. 1G). They occur in the south-west of France, the Atlantic regions as well as the south of Scandinavia. Currently, all these subspecies and form are considered as B. sylvarum (Rasmont et al., 2015a;[START_REF] Williams | An annoted checklist of bumble bees with an analysis of patterns of description (Hymenoptera: Apidae, Bombini)[END_REF].

All other species included in this paper form a monophyletic group with B. ruderarius and B. sylvarum [START_REF] Cameron | A comprehensive phylogeny of the bumble bees (Bombus)[END_REF] and will be treated as comparison group.

Bombus inexspectatus [START_REF] Tkalců | Eine neue Hummel-Art der Gattung Agrobombus Vogt aus dem Alpengebiet[END_REF] (Fig. 1A) is a sub-alpine uncommon European species restricted in the Cantabrian Mountains and the Alps from 1200m to 2100m in altitude [START_REF] Amiet | Hymenoptera Apidae, 1. Teil: Allgemeiner Teil, Gattungsschlüssel, die Gattungen Apis, Bombus und Psithyrus[END_REF]Rasmont et al., 2015a;[START_REF] Tkalců | Eine neue Hummel-Art der Gattung Agrobombus Vogt aus dem Alpengebiet[END_REF][START_REF] Tkalců | Über Agrobombus inexspectatus Tkalců[END_REF][START_REF] Yarrow | Is Bombus inexspectatus (Tkalcu) a workless parasite?[END_REF]. It has been described as a species adapted to parasitic life [START_REF] Müller | A scientific note on Bombus inexspectatus (Tkalcu, 1963): evidence for a social parasitic mode of life[END_REF][START_REF] Yarrow | Is Bombus inexspectatus (Tkalcu) a workless parasite?[END_REF] with B. ruderarius as its only host [START_REF] Müller | A scientific note on Bombus inexspectatus (Tkalcu, 1963): evidence for a social parasitic mode of life[END_REF]. There is no doubt about its specific status.

B. mlokosievitzii Radoszkowski,1877 is a ponto-mediterranean species whose distribution extends from the Balkan Mountains to Caucasian region via the south of the Black Sea [START_REF] Rasmont | Atlas of the European Bees: genus Bombus[END_REF]. Three subspecies diagnosable by their coat color variation has been defined: (i) B. mlokosievitzii mlokosievitzii Radoszkowski, 1877 with a wide greyish white collare and scutellare bordering a black interalar band. This subspecies is distributed in the north of Anatolia; (ii) B. mlokosievitzii convergens [START_REF] Skorikov | Novyya formy shmeley (Hymenoptera, Bombidae). (Predvaritel'nye diagnozy). I[END_REF] characterized by a thorax completely greyish white is distributed in the Caucasian region and (iii) B. mlokosievitzii vogtiellus diagnosable by their thorax completely black (Fig. 1B) and distributed in Greece, Macedonia, Bulgaria and west of Anatolia. The presence of a reddish band on tergite 2 is common to all subspecies. Unfortunately we were not able to collect all subspecies inside this taxon and only the subspecies mlokosievitzii was considered in this study. [START_REF] Skorikov | Les formes nouvelles des bourdons (Hymenoptera, Bombidae)[END_REF] is restricted to the north east of Anatolia and the Caucasus region. It is an uncommon species with two greyish bands on the thorax and one on the tergite 1, the rest of the abdomen is yellowish (Fig. 1H). There is no doubt about its specific status.

B. velox

B. veteranus [START_REF] Fabricius | Entomologica systematica emendata at aucta. Secundum classes, ordines, genera, species adjectis synonimis[END_REF] lives in sparse population in the plains of northern Europe [START_REF] Rasmont | Atlas of the European Bees: genus Bombus[END_REF]. This species presents very little color variation and is characterize by a yellowish coat color on all the body except the presence of a black interalar band (Fig. 1I). There is no doubt about its specific status. Sampling 150 male and female specimens were collected between 2001 and 2017 in the Palaearctic region (supplementary file 1, Tab. 1). All specimens were determined based on their morphology. We attributed a name to the specimens without taxonomic a priori on the species status. The ingroup comprises: ruderarius from Estonia (n=5), France (n=16), Macedonia (n=1), Serbia (n=1), Slovakia (n=4), Sweden (n=1) and Switzerland (n=5), montanus from Andorra (n=3), Spain (n=2) and France (n=5), simulatilis from Iran (n=3) and Turkey (n=9), sartus from Kyrgyzstan (n=2) and Mongolia (n=2), sylvarum from Belgium (n=3), France (=13),Germany (n=4),Italy (n=3),Poland (n=6),Serbia (n=2),Slovakia (n=1),Spain (n=2) and Sweden (n=4), daghestanicus from Iran (n=1) and Turkey (n=8), nigrescens from Spain (n=1) and Sweden (n=5), rogenhoferi from Italy (n=2). The comparison group comprises: inexspectatus from Italy (n=9); mlokosievitzii from Turkey (n=11), velox from Turkey (n=1) and veteranus from Belgium (n=4), France (n=4) and the Netherlands (n=4). The phylogenetically related species B. mucidus (n=3) was used as outgroup.

Genetic trait analyses

Two genes commonly used in bumblebee phylogeny were sequenced [START_REF] Cameron | A comprehensive phylogeny of the bumble bees (Bombus)[END_REF][START_REF] Martinet | Following the cold: geographical differentiation between interglacial refugia and speciation in the arcto-alpine species complex Bombus monticola (hymenoptera: Apidae)[END_REF][START_REF] Williams | Unveiling cryptic species of the bumblebee subgenus Bombus s. str. worldwide with COI barcodes (Hymenoptera: Apidae)[END_REF]: the "barcode" region of the cytochrome oxidase 1 (COI) mitochondrial gene was sequenced from 88 specimens, and the nuclear protein-coding gene phosphoenolpyruvate carboxykinase (PEPCK) was sequenced from 83 specimens (supplementary file 1).

We carried out polymerase chain reaction amplifications with new design primer pairs CSylG-F/CSylG-R:

(5'-TGATCAGGAATAATTGGATCTTCA-3') / (5'-TGATCAGGAATAATTGGATCTTCA-3') for COI and PSylG-F/PSylG-R: (5'-CTTTAAAGGCAATTGTATCAAAGTT-3') / (5'-CTCGCCAATCGGTGATCTAT-3') for PEPCK. We performed sequencing procedures described in [START_REF] Lecocq | Scent of a break-up: phylogeography and reproductive trait divergences in the red-tailed bumblebee (Bombus lapidarius)[END_REF]. Sequences were aligned with BioEdit version 7.2.5 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. The resulting COI (624bp) and PEPCK (894bp) sequences are available on GenBank (accession numbers in supplementary file 1).

We investigated the potential genetic differentiation for each gene individually within the six taxa through haplotype networks analyses. We used the median-joining method with Network 4.6.1.1 software (www.fluxus-engineering.com) to produce haplotype networks. We weighted transversions twice as high as transitions to reconstruct the network (Lecocq et al., 2015a;[START_REF] Martinet | Following the cold: geographical differentiation between interglacial refugia and speciation in the arcto-alpine species complex Bombus monticola (hymenoptera: Apidae)[END_REF]. In phylogenetic analyses, gaps (deletion or insertion) are usually treated as missing data. Because of the presence of gaps sites in our PEPCK genes, two haplotype networks considering or not the gaps sites will be performed in order to be aware of phylogenetic information provide by these two methods.

In phylogenetic analyses, we analyzed each gene independently with maximum likelihood (ML) and Bayesian (MB) methods to explore the genetic divergence and define lineages within the different groups. For ML methods, we partitioned each gene to explore the best substitution model: i) PEPCK into two introns and one exon; ii) COI and PEPCK exons by base position (1 st , 2 nd and 3 rd ) and choose the best fitting substitution models with JModelTest Server 2.0 [START_REF] Posada | jModelTest: Phylogenetic model averaging[END_REF] using the Akaike information criterion corrected for small sample sizes [START_REF] Hurvich | Regression and time series model selection in small samples[END_REF]. The chosen models were: i) For COI: TIM1 + I (1 st ), HKY

(2 nd ), and TPM3uf + I (3 rd ); ii) for PEPCK, exon: F81 (1 st ), JC (2 nd ), K80 (3 rd ); intron 1: TMP3uf + I; intron 2: HKY. Selected models which are not implemented in MrBayes were substituted by the closest over-parameterized model. For each gene, ML analyses were performed with ten independent runs in Garli 2.0 [START_REF] Zwickl | Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criteria[END_REF]; the topology and -ln L was the same among replicates. The run with the highest likelihood was retained. Statistical significance in nodes was evaluated with 10000 non-parametric bootstrap replicates.

Topologies with bootstrap values ≥85% were considered as well supported [START_REF] Hillis | An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis[END_REF]. For MB method, we performed Bayesian inference analyses with mrbayes 3.1.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF]. Ten independent analyses were achieved for each gene (100 million generations, four chains with mixed models, default priors, saving trees every 1000 generations). Then the first 10 million generations were removed as burn-in procedure. A majority-rule 50% consensus tree was constructed. We considered as statistically significant only branch supports (topologies) with high posterior probabilities (≥0.95) [START_REF] Wilcox | Phylogenetic relationships of the dwarf boas and a comparison of Bayesian and bootstrap measures of phylogenetic support[END_REF]. The phylogenetic analysis based on PEPCK gene fragment considered gaps as missing data. All trees were rooted with the taxon B. mucidus.

In order to recognize species threshold, we applied the Bayesian implementation of the general mixed Yule-coalescent (bGMYC) method to the COI dataset by using R Package "bGMYC" [START_REF] Reid | Phylogenetic estimation error can decrease the accuracy of species delimitation: A Bayesian implementation of the general mixed Yulecoalescent model[END_REF]. A range of probabilities < 0.05 was considered as strong evidence that taxa was heterospecific while a range of probabilities > 0.95 suggested that taxa was conspecific [START_REF] Reid | Phylogenetic estimation error can decrease the accuracy of species delimitation: A Bayesian implementation of the general mixed Yulecoalescent model[END_REF]. BEAST 1.7.4 [START_REF] Drummond | Bayesian phylogenetics with BEAUTI and BEAST 1.7[END_REF] was used to generate ultrametric trees with a phylogenetic clock model in order to calculate a posterior distribution of trees (length of the MCMC chain: 1 billion generations). The first million sampled trees were burned-in using the maximum clade credibility method and setting the posterior probability limit to 0. The bGMYC analysis was based on 1000 trees sampled every 10 000 generations. For each of these 1000 trees, the MCMC was made of 100 000 generations, discarding the first 90 000 as burn-in and sampling every 100 generations.

Eco-chemical trait analyses

We focused on the cephalic labial gland secretions (CLGS) of males, the most commonly reproductive trait involved in the bumblebee pre-mating recognition [START_REF] Ayasse | Chemical ecology of bumble bees[END_REF].

Currently, the composition of the CLGS is known for 15 species of Thoracobombus [START_REF] Ayasse | Chemical ecology of bumble bees[END_REF][START_REF] Brasero | First Chemical Analysis and Characterization of the Male Species-Specific Cephalic Labial-Gland Secretions of South American Bumblebees[END_REF]Brasero et al., 2018a,b) and this study will increase the knowledge inside this group to 17 species. These secretions are species-specific [START_REF] Calam | Species and Sex-specific Compounds from the Heads of Male Bumblebees (Bombus spp.)[END_REF] and provide an efficient diagnostic characters for species delimitation [START_REF] Brasero | First Chemical Analysis and Characterization of the Male Species-Specific Cephalic Labial-Gland Secretions of South American Bumblebees[END_REF]. They are complex mixtures of mainly aliphatic compounds synthesized de novo [START_REF] Coppée | Intraspecific variation of the cephalic labial gland secretions in Bombus terrestris (L.) (Hymenoptera: Apidae)[END_REF][START_REF] Žáček | De Novo Biosynthesis of Sexual Pheromone in the Labial Gland of Bumblebee Males[END_REF] in the head of bumblebee males. Main components were defined as those with the highest relative amount in at least one individual of the taxon.

All specimens were killed by freezing at -20°C and the CLGS were extracted with 400 µl of heptane (method described in De Meulemeester, Gerbaux, Boulvin, [START_REF] De Meulemeester | A simplified protocol for bumble bee species identification by cephalic secretion analysis[END_REF]. Samples were stored at -40°C prior to analysis. We were able the sample 111 specimens belonging to the six studied taxa (Tab. 1).

We determined the CLGS composition by gas chromatography-mass spectrometry (GC/MS) using a Focus GC (Thermo Scientific) with a non-polar DB-5 ms capillary column [5% phenyl (methyl) polysiloxane stationary phase; column length 30 m; inner diameter 0.25 mm; film thickness 0.25 μm] coupled to DSQ II quadrupole mass analyzer (Thermo Scientific)

with 70 eV electron impact ionization. We used a splitless injection mode (220°C) and helium as a carrier gas (one ml/min). The temperature program of the oven was set to 70°C

for two minutes and then heated up at a rate of 10°C/min to 320°C. The temperature was then held at 320°C for five minutes. Compounds were identified in Xcalibur TM using the retention times (tr) and mass spectra of each peak, in comparison to those at National Institute of Standards and Technology library (NIST, U.S.A). Double bond positions (C=C) were determined by dimethyl disulfide (DMDS) derivatization [START_REF] Cvacka | Unusual fatty acids in the fat body of the early nesting bumblebee, Bombus pratorum[END_REF].

The CLGS compounds of all samples were quantified by a gas chromatograph-flame ionization detector Shimadzu GC-2010 with a SLB-5ms non-polar capillary column (5% phenyl (methyl) polysiloxane stationary phase; 30-m column length; 0.25-mm inner diameter; 0.25-µm film thickness) with the same chromatographic conditions as in GC/MS. Peak areas of compounds were detected in GCsolution Postrun (Shimadzu Corporation) with automatic peak detection and noise measurement. We calculated relative amounts (RA in %) of compounds in each sample by dividing the peak areas of compounds by the total area of compounds. We discarded all compounds for which RA were recorded as less than 0.1% for all specimens [START_REF] De Meulemeester | A simplified protocol for bumble bee species identification by cephalic secretion analysis[END_REF]. The data matrix (supplementary file 2) for each taxon was based on the alignment of each relative proportion of compound between all samples performed with GCAligner 1.0 (Dellicour & Lecocq, 2013).

Before each samples injection, a standard (Kovats) containing a mix of hydrocarbons (alkanes) from C10 (decane) to C40 (tetracontane) was injected in order to facilitate the alignment and the identification of compounds. Kovats indices were calculated with GC Kovats 1.0 (Dellicour & Lecocq, 2013).

Clustering method were performed using R 3.3.2 (R Development Core Team, 2016) to detected CLGS differentiations between taxa. A Canberra distance matrix based on CLGS (RA of each compound) was computed. An unweighted pair group method with arithmetic mean (UPGMA) was used as clustering method (R-package ape, [START_REF] Suzuki | Pvclust: Hierarchical Clustering with P-Values via Multiscale Bootstrap Resampling[END_REF]. We assessed the uncertainty in hierarchical cluster analysis using p-values calculated via multiscale bootstrap resampling with 50 000 bootstrap replications (significant branch support > 0.85) (R-package pvclust, [START_REF] Suzuki | Pvclust: Hierarchical Clustering with P-Values via Multiscale Bootstrap Resampling[END_REF]. We assessed CLGS differentiations between taxa by performing a permutation multivariate analysis of variance using distance matrix (perMANOVA) (R package vegan; [START_REF] Oksanen | Vegan: Community Ecology Package. Tertiary Vegan: Community Ecology Package[END_REF]. When a significant difference was detected, we performed a pairwise multiple comparison with an adjustment of P-values (Bonferroni correction) to avoid type I errors.

To determine the indicator compounds (IC) of each taxon, we used the indicator value (IndVal) method [START_REF] Claudet | Assessing the effects of marine protected area (MPA) on a reef fish assemblage in a northwestern Mediterranean marine reserve: Identifying community-based indicators[END_REF][START_REF] Dufrêne | Species assemblages and indicator species: The need for a flexible asymmetrical approach[END_REF]. The value given is the product of relative abundance and relative frequency of occurrence of a compound within a group. We evaluated the statistical significance of a compound as an indicator at the 0.01 level with a randomization procedure.

Decision framework of taxonomic status

We used an approach that corresponds to the strictest commonly used framework called "integration by congruence" by [START_REF] Padial | The integrative future of taxonomy[END_REF]. We thus assigned the specific status to a taxon (i) genetically differentiated in all genetic markers (i.e. unique haplotypes), (ii) which constitute a monophyletic group with high branch support, and (iii) with a significant differentiation in CLGS composition (significance differentiation in perMANOVA and high bootstrap values > 0.85). All criteria used must be convergent to assign specific status. This approach can lead to underestimation of species differentiation but reduces the taxonomic inflation. We assigned the subspecies status to phenotypically distinct allopatric population that does not diverge in all lines of evidence. This decision reduces the 'underestimate's risk' of our strict integrative approach by naming them as a subspecies [START_REF] Ennen | Clinal variation or validation of a subspecies? A case study of the Graptemys nigrinoda complex (Testudines: Emydidae)[END_REF][START_REF] Hawlitschek | Island evolution and systematic revision of comoran snakes: Why and when subspecies still make sense[END_REF].

Results

Genetic trait analyses

All phylogenetic analyses (ML and MB) showed the expected differentiation between outgroup and ingroup (Fig. 2A,3A). Seven strongly supported (bootstrap>0.95) monophyletic groups are detected in our COI phylogenetic analyses (Fig. 2A Iran. As expected, we found a less structured tree in the PEPCK gene fragment for all taxa.

PEPCK gene analyses showed little phylogenetic information and failed to reconstruct the phylogenetic affinities between all taxa (Fig. 3A). However, we found some strongly supported groups as inexspectatus and mlokosievitzii. As for COI gene, no geographical entities were underlined by our PEPCK phylogenetic analyses expected for "ruderarius group" where the unexpected lineage simulatilis was revealed and strongly supported in our MB (Fig. 3A) but not in our ML analysis.

15 private haplotypes were highlighted for COI gene while 11 and 10 haplotypes were highlighted in PEPCK genes respectively considering or not the gaps sites by our network analysis (Fig. 2B,3B,C). Within the "ruderarius and sylvarum groups", only simulatilis displayed specific haplotypes in COI and PEPCK, while daghestanicus had specific COI haplotype (Fig. 2B,3B,C). Considering all taxa, COI revealed 72/624 nucleotide sites substitution corresponding to 11.54% of phylogenetic information. Compared to the taxa inside their groups, simulatilis and daghestanicus revealed respectively 0.96% (6/624 diagnostic nucleotide sites) and 0.64% (4/624 diagnostic nucleotide sites) of phylogenetic information in COI gene (Fig. 2B). Considering all taxa, PEPCK gene revealed 143/894 (considering gaps) and 23/894 (not considering gaps) nucleotide sites respectively corresponding to 16% and 2.6% of phylogenetic information. Compared to the taxa inside their groups, simulatilis revealed 1.79% (16/894 diagnostic nucleotide sites) of phylogenetic information when gaps are considered (Fig. 3C) and 0.34% (3/894 diagnostic nucleotide sites) when gaps are not considered (Fig. 3B).

The tree generated by bGMYC analysis highlighted several entities with low probabilities (<0.05) to be conspecific with the other ones (Fig. 4). It suggested the delimitation of 7

prospective species (P<0.05) (Fig. 4) which match with those obtained in the COI tree (ML and MB, Fig. 2A

Eco-chemical trait analyses

The chemical analyses highlighted 7 strongly supported groups (bootstrap > 85%) (Fig. 5). A: from G; see supplementary file 2). For each group, we detected main compounds, A:

velox (
octadecen-1-ol (88.2%); B: hexadec-7-en-1-ol (17.2-57.8%), octadec-11-en-1-ol (12.2-32.6%) and octadec-11-enyl acetate (10-23.4%); C: hexadec-7-en-1-ol (14-46.7%) and hexadec-7enyl acetate (19.6-45.9%); D: hexadec-7-en-1-ol (2.2-57.5%) and hexadec-7-enyl acetate (20.1-66.6%); E: hexadec-9-en-1-ol (1.1-57.8%) and ethyl octadec-11-enoate (1.5-31.4%); F:

hexadec-9-en-1-ol (0-62.3%), hexadec-9-enyl acetate (0-32.7%), tricos-9-ene + tricos-7-ene

(2.1-59.2%) and hexadecenyl hexadecenoate2 (0.1-25.1%) and G: hexadec-9-en-1-ol (4.5-59.9%), octadecadien-1-ol (0.4-22%), and tricos-9-ene + tricos-7-ene (5.7-54.2%) and tricosane (2-20%). Moreover, the IndVal method highlighted several significantly indicator compounds (IC) (IndVal value>70) for the group A (IC=4), B (IC=11), C (IC=3), D (IC=7), E (IC= 5), F (IC=1) and G (IC=0) (supplementary file 2). Despite the separation we observed between montanus + ruderarius and simulatilis our IndVal method did not find indicator compounds for simulatilis, however, our statistical analysis significantly supports this differentiation (bootstrap > 85%). perMANOVA test confirmed the differentiations between all groups (see supplementary file 3) and particularly between montanus + ruderarius and simulatilis (p-value: 0.041; F: 2.9986).

Decision framework of taxonomic status

Within the "ruderarius group" (ruderarius, sartus, montanus and simulatilis), operational criteria did not reflect the current subspecific differentiation (Tab. 2). CLGS and haplotype networks analyses detected differentiation between simulatilis and the other taxa. We then considered B. ruderarius simulatilis as a subspecies inside the "ruderarius group". This subspecies occurs in mountainous regions of Anatolia, the Caucasus region as well in north

Iran. We also assigned the subspecific status B. ruderarius ruderarius [START_REF] Müller | Zoologiae Danicae prodromus, seu animalium Daniae et Norvegiae indigenarum characteres, nomina, et synonyma imprimis popularium[END_REF] to the other taxa (Tab. 2).

The genetic structure observed in COI, PEPCK and CLGS did not reflect the current subspecific status encountered in the "sylvarum group" (sylvarum, daghestanicus, nigrescens and rogenhoferi). Based on its unique haplotype in COI we decided to consider B. sylvarum daghestanicus as a subspecies inside the "sylvarum group" as described by Radoszkowski (1877). This subspecies occurred in mountainous regions of Anatolia, the Caucasus region as well in Iran. We then assigned the subspecific status B. sylvarum sylvarum [START_REF] Müller | Zoologiae Danicae prodromus, seu animalium Daniae et Norvegiae indigenarum characteres, nomina, et synonyma imprimis popularium[END_REF] to the other taxa inside which we considered the forms sylvarum, nigrescens and rogenhoferi based on their color patterns (Tab. 2). The specific status of B. inexspectatus, B. mlokosievitzii, B. velox and B. veteranus were confirmed by our results.

Discussion

Genetic character along with CLGS can confirm with a high degree of certainty the speciation processes between taxa. We established the species status of our different taxa according to their divergences in genetic markers (mitochondrial and nuclear) and their CLGS differentiation (Tab. 2).

Our integrative approach support the specific status of all closely related consubgeneric six species studied here [START_REF] Cameron | A comprehensive phylogeny of the bumble bees (Bombus)[END_REF][START_REF] Williams | An annoted checklist of bumble bees with an analysis of patterns of description (Hymenoptera: Apidae, Bombini)[END_REF]. Similar interspecific differentiations were recorded independently based on DNA [START_REF] Cameron | A comprehensive phylogeny of the bumble bees (Bombus)[END_REF][START_REF] Hines | The phylogenetic position of the bumble bee inquiline Bombus inexspectatus and implications for the evolution of social parasitism[END_REF] and CLGS composition [START_REF] Appelgren | Marking pheromones of Megabombus bumble bee males[END_REF]Brasero et al., 2018a;[START_REF] Terzo | Intra and interspecific variability of the cephalic labial glands' secretions in male bumblebees: the case of Bombus (Thoracobombus) ruderarius and B. (Thoracobombus) sylvarum Hymenoptera, Apidae[END_REF]. However, the analyses in this study were carried out on a larger sampling covering the distribution of the species. Within "ruderarius group" and "sylvarum group" and despite some phenotypic differentiations, all studied taxa are regarded as conspecific (Tab. 2). ruderarius and sylvarum groups appear respectively as single species with high geographic phenotypic differentiations. In the light of our results, we decide for the "sylvarum group" to use the oldest available nominal species name as the valid name for all of them: B. sylvarum sylvarum except for daghestanicus where we maintain the . inexspectatus,B. mlokosievitzii,B. veteranus and B. velox,our analyses (Fig 2A,3A,4 & 5) confirm clear differentiation between these species [START_REF] Cameron | A comprehensive phylogeny of the bumble bees (Bombus)[END_REF][START_REF] Hines | The phylogenetic position of the bumble bee inquiline Bombus inexspectatus and implications for the evolution of social parasitism[END_REF] (Rasmont et al., 2015a). Divergence of color patterns (sometimes very pronounced: see the melanic form nigrescens), seems to be a simple intraspecific variation as encountered in other bumblebees species such as B. pascuorum (Lecocq, Brasero, Martinet, Valterova & Rasmont, 2015c). However, even if unique haplotypes (Fig. 2B) are found in daghestanicus (COI gene), there were not strongly supported as reciprocally monophyletic group in our phylogeny (Fig. 2A). No private haplotype was found in PEPCK gene for this taxon (Fig. 3B,C). Moreover, inside the "sylvarum group", high probabilities (>0.05) to be conspecific is showed by the bGMYC analyses (Fig. 4). On the other side, the CLGS did not highlight any divergence between the different populations which would imply that all of them could recognize each other as sexual partners.

B. ruderarius is also a widespread species and its distribution is almost identical to that of B.

sylvarum. However, B. ruderarius is absent from Sicily and the southern Italy but the subspecies B. ruderarius tunensis occurs in the east of Algeria and Tunisia (Rasmont et al., 2015a). The same configuration as in B. sylvarum has been highlighted. Indeed, the divergence in color patterns seems to be a simple intraspecific variation and no diagnostic character was found to discriminate significantly the different population inside B. ruderarius.

The unique haplotypes found in COI and PEPCK (Fig. 2B, 3B, C) for simulatilis were not strongly supported as reciprocally monophyletic group in our phylogenies (Fig. 2A, 3A) as well in our bGMYC analyses (Fig. 4). However, the CLGS of this Turkish population differ from other populations and it is well supported in our cluster analysis (Fig. 5) but share main compounds (hexadec-9-en-1-ol, tricos-9-ene + tricos-7-ene) with the other B. ruderarius taxa (supplementary file 2). We can therefore hypothesize that this divergence involved in the recognition system is not sufficient to induce a pre-mating reproductive barrier. This geographic differentiation sharing main compounds could suggest only a dialect between populations as already described in moths [START_REF] Tòth | Attraction of male turnip moths Agrotis segetum (Lepidoptera: Noctuidae) to sex pheromone components and their mixtures at 11 sites in Europe, Asia, and Africa[END_REF] and solitary bees [START_REF] Vereecken | Population differentiation in female sex pheromone and male preferences in a solitary bee[END_REF]. Although the taxon simulatilis seems more advanced on the path of speciation than daghestanicus, complementary tests based on ethological behavior are needed.

We then hypothesize that these results could reflect the recent divergence of these taxa.

Unfortunately, we have not been able to analyze the subspecies citrinofasciatus and tunensis respectively inside the "sylvarum group" and the "ruderarius group".

Turkey is divided by a diagonal belt which is a massive structure formed by continuous mountains (Miocene-Pliocene) from the north-east towards the south-west. These mountains isolated populations from east and west in many species of insects or plants [START_REF] Çiplak | Distribution of Orthoptera in Relation to the Anatolian Diagonal in Turkey[END_REF][START_REF] Çiplak | Distribution of Tettigoniinae (Orthoptera, Tettigoniidae) bush-crickets in Turkey: the importance of the Anatolian Taurus Mountains in biodiversity and implications for conservation[END_REF][START_REF] Ekim | The Anatolian Diagonal: fact or fiction?[END_REF]. Moreover, the distribution of taxa from the West of this barrier generally spread through Europe while the taxa from the east spread through Iran and Asia. In addition with this geographical particularities, climatic change during the ice ages pushed many northern populations especially those found in the Balkans and Caucasus to the south and led to their isolation and speciation at higher altitudes [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF][START_REF] Hewitt | The genetic legacy of the quaternary ice ages[END_REF]. These two taxa (B. ruderarius simulatilis and B. sylvarum brodmannicus (Pyrobombus) and B. niveatus (Sibiricobombus) [START_REF] Rasmont | Atlas of the European Bees: genus Bombus[END_REF].

Consider GAPS or not as phylogenetic information?

Our PEPCK gene fragment contains gaps sites (representing an insertion or deletion and also called "indels") and our statistical method for phylogenetic estimation (ML and MB) consider the gap as missing data. Indeed, in most of taxonomic studies, the indels are treated as missing data as this is the case for birds [START_REF] Alström | Integrative taxonomy of the Russet Bush Warbler Locustella mandelli complex reveals a new species from central China[END_REF][START_REF] Mckay | Oncorporating color into integrative taxonomy: analysis of the varied tit (Sittiparus varius) complex in East Asia[END_REF], frogs [START_REF] Glaw | Integrative taxonomy of Malagasy treefrogs: combination of molecular genetics, bioacoustics and comparative morphology reveals twelve additional species of Boophis[END_REF], mammals [START_REF] Ndiaye | Integrative taxonomy of a poorly known Sahelian rodent, Gerbillus nancillus (Muridae, Gerbillinae)[END_REF], spiders [START_REF] Rezac | Integrative taxonomy and evolutionary history of a newly revealed spider Dysdera ninnii complex (Areneae: Dysderidae)[END_REF]. Considered indels will increase the phylogenetic information even if they are located in introns (in our case, all indels are located in the first introns of the PEPCK gene fragment). These gaps reflect the evolutionary history of the taxa and thus contain phylogenetic information which could be very informative particularly for simulatilis where the divergence estimation with all other ruderarius populations remains unclear. It's seems therefore that the treatment of indels has not been fully investigated in taxonomic studies and delimitaion of species. Moreover, in our study, haplotype network analysis considering or not the indels do not bring us any additional information except for B.

velox which not differs from B. ruderarius if we not considered indels in our analysis (Fig. 3B). Indeed, only gap sites allow to separate B. velox from B. ruderarius. However, both species are respectively strongly differenciated by the genitalia and other morphological characters.

Conservation implication

Our integrative approach brings to attention the relevance of geographically isolated groups differentiated in genetic and reproductive traits. This strict approach allows defining taxonomic status (species or subspecies) and providing a decision framework for policymakers and conservation organizations. Except for B. inexspectatus [START_REF] Rasmont | Bombus inexspectatus. The IUCN Red List of Threatened Species 2015[END_REF] considered as endangered species in the International Union for Conservation of Nature

  subspecific status B. sylvarum daghestanicus from east of Turkey and Iran because of its private haplotype in COI. Inside the "ruderarius group" we decide to conserve the subspecific status B. ruderarius simulatilis for the population in the east of Turkey and Iran based on their CLGS differentiation and private haplotype (COI and PEPCK) and raised the other populations to the oldest available nominal species name B. ruderarius ruderarius (see decision framework Tab. 2). For the comparison groups B

  daghestanicus) are known to occur in the east part of the diagonal belt and may have colonized Turkey from Caucasus and Asia while the taxa to the west of this barrier may have colonized Turkey from Balkans. It is also interesting to note that B. sylvarum daghestanicus and B. ruderarius simulatilis are characterized by the same color pattern (white bands and red tail) (Fig. 1D, E) which could be explained by a local Müllerian mimicry processes (Hines & Williams, 2012). Other species from other subgenus also posses the same color pattern in this region and could confirm this process: B. cullumanus apollineus (Cullumanobombus), B. sicheli cazuroi and B. incertus (Melanobombus), B. handlirschianus (Mendacibombus), B.

(FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. A: Majority rule (50%) consensus tree based on maximum likelihood analyses of

Figure 4 .

 4 Figure 4. bGMYC results based on COI phylogenetic tree. The vertical and horizontal lines

  P=1) and (vii) one group with the outgroup B. mucidus (P=1). The pairwise matrix shows a non significant heterospecificity threshold between simulatilis and the other taxa from ruderarius group (P>0.05).

	): (i); one group with inexspectatus specimens (P=1); (ii); one group with
	velox specimens (P=1); (iii) one group with ruderarius, sartus, montanus and simulatilis

specimens (P>0.16-1); (iv) one group with veteranus specimens (P=1); (v) one group including sylvarum, daghestanicus, nigrescens, rogenhoferi specimens (P>0.34-1); (vi) one group with mlokosievitzii specimens (

  . Inside B. mlokosievitzii, we have not been able to analyze the subspecies convergens and vogtiellus. Intraspecific differentiation of B. sylvarum and B. ruderarius B. sylvarum is a widespread species and can be found in Central Spain, Sicily, southern Italy, Greece and Southern Turkey of its distribution. It reaches Ireland and northem Portugal to the West and Mongolia to the East. To the north, this species nearly reaches the Arctic Circle

Acknowledgments

The authors thank the Parco Nazionale dei Monti Sibillini for granting permission to collect in their respective territories to Paolo Biella. Special thanks go to A. Cetkovic and A. Popovic (University of Belgrade), R. De Jonghe, P. Sagot and V. Cyriaque (University of Mons) for their help in the sampling but also to Simon Dellicour (University of Leuven) for his advices in phylogenetic analyses. Computational resources have been provided by the Consortium des Equipements de Calcul Intensif (CECI), funded by the Belgian FRS-FNRS (Fonds de la Recherche Scientifique). BM contributes as a PhD student granted by the Research Council of University of Mons and by the FRS-FNRS. Part of this work (Eco-chemical trait differentiation) was supported by the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic (#61388963). The research has received funding from the FRS-FNRS (Fonds de la Recherche Scientifique) (2013/V 3/5/307-IB/JN-11888) and the European Community's Seventh Framework Program, STEP Project (Status and Trends of European Pollinators, www.step-project .net, grant agreement no 244090, FP7/2007-2013).

This work was supported by the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic under grant number 61388963; the FRS-FNRS (Fonds de la Recherche Scientifique) under grant number 2013/V 3/5/307-IB/JN-11888 and the European Community's Seventh Framework Program, STEP Project (Status and Trends of European Pollinators, www.step-project .net, under grant number 244090, FP7/2007-2013).

probabilities. B: Median-joining network of haplotypes based on cytochrome oxidase 1 (COI) sequences. Numbers on the lines correspond to the number of mutation between haplotypes.


Dino Ienco
email: dino.ienco@irstea.fr

Yoann Pitarch
email: yoann.pitarch@liris.cnrs.fr

Pascal Poncelet
email: pascal.poncelet@lirmm.fr

Maguelonne Teisseire
email: maguelonne.teisseire@irstea.fr

Knowledge-free Table Summarization

Considering relational tables as the object of analysis, methods to summarize them can help the analyst to have a starting point to explore the data. Typically, table summarization aims at producing an informative data summary through the use of metadata supplied by attribute taxonomies. Nevertheless, such a hierarchical knowledge is not always available or may even be inadequate when existing. To overcome these limitations, we propose a new framework, named cTabSum, to automatically generate attribute value taxonomies and directly perform table summarization based on its own content. Our innovative approach considers a relational table as input and proceeds in a two-step way. First, a taxonomy for each attribute is extracted. Second, a new table summarization algorithm exploits the automatic generated taxonomies. An information theory measure is used to guide the summarization process. Associated with the new algorithm we also develop a prototype. Interestingly, our prototype incorporates some additional features to help the user familiarizing with the data: (i) the resulting summarized table produced by cTabSum can be used as recommended starting point to browse the data; (ii) some very easy-to-understand charts allow to visualize how taxonomies have been so built; (iii) finally, standard OLAP operators, i.e. drill-down and roll-up, have been implemented to easily navigate within the data set. In addition we also supply an objective evaluation of our table summarization strategy over real data.

Introduction

Nowadays, modern technologies allow to collect huge amount of raw but potentially very knowledgable data which are available at a very low level of granularity, e.g, sensor data, web logs. When facing this very detailed information the domain expert might experience big trouble determining from where he/she can start exploring these data. Actually, this data exploration is typically the first step in any analysis process. Nevertheless, when the available quantity of data is too abundant or the user is not so expert in the domain, this first step is often problematic and automatic approaches can help to obtain some insights about the way to effectively explore the data [?]. For instance, Explorative Data Mining (EDM) tools help the user in preliminary analysis giving him/her the possibility to have a first meaningful view on the data. In this direction, it should be noted that two orthogonal research problems might coexist. Assuming a relational table setting, i.e., lines correspond to tuples whereas rows correspond to attributes/dimensions, the former problem is related to deal with the curse of dimensionality by the means of dimensionality reduction or aggregation [?], whereas the latter problem deals with reducing the number of tuples while providing the user with still a very representative view of his/her data. In this paper, we address the latter, i.e., the tuple reduction problem.

One way to manually deal with this tuple reduction problem is using data warehouses coupled with OLAP engines [?]. These interactive tools allow the analyst to aggregate and navigate through data by the mean of the drill-down and roll-up operators that exploit, when available, the attribute hierarchies/taxonomies4 . OLAP engines demand human supervision while an automatic way for addressing this problem is supplied by the table summarization process (TSP) [?].

The table summarization process (TSP) provides an aggregated representation of a relation table and thus helps the user familiarizing with the data. Typically, TSP is particularly well adapted to the rapid development of mobile devices, e.g., smart phones or tablets, since it enables visualizing data respecting the screen size constraints imposed by the new devices generation [?]. To cope with this issue, TSP can be exploited from smartphone devices to supply synopsis (or summary) of relational data. In this work we focus our research on the second issue and thus propose a new innovative table summarization algorithm.

Our work is essentially motivated by the following observation: data often lacks of associated metadata information, e.g., attribute taxonomies. This lack drastically limits the panel of possible approaches (OLAP, TSP) to use to reduce the number of tuples. Indeed, both OLAP and existing TSP techniques assume attribute hierarchies to be available. This assumption is clearly a strong constraint because most of the times such a hierarchical knowledge is not available or may even be inadequate when existing. On the other hand, using inappropriate hierarchy can provide useless results since they can be too general and bias the OLAP and TSP results. Thus, proposing techniques that are able to meaningfully summarize huge quantities of data without leveraging any other knowledge than the one provided by the data might be very useful for analysis purpose.

Contrary to the previous techniques, our framework, called cTabSum, relaxes this constraint producing table summaries without needing any additional information. The main advantage of cTabSum lies in its ability to automatically generate contextual Attribute Values Taxonomies (cAVTs) and to use them to automatically summarize the data. This original feature also allows our approach to be used over relation tables where metadata information are too general or do not fit the domain of investigation. An example of application can be represented by the visualization, over mobile device, of query results. cTabSum can automatically aggregate query results exploiting data dependency. After the performed aggregation, the summarized table can be easily visualized on a smartphone screen. The final result of cTabSum is a summarized table that can be considered as a recommended aggregation of the data. It can also be considered as an entry point from which the user can start browsing the data using drill-down and roll-up operators. The main contributions of our work are the following:

-A general table summarization approach that can be applied over data table where no metadata are available or metadata are too general for the considered domain; -The final result can be exploited as a starting point to browse and navigate through the data; -A prototype that integrates the cTabSum algorithm in an interactive and easy-to-use environment. Drill-Down and Roll-up operators are implemented to navigate data starting from the recommended summarization.

The rest of the paper is organized as follows. Section 2 presents the related work and positions our contributions w.r.t. the state of the art. In Section 3 we describe our proposal to produce TSP without any kind of metadata information. An experimental evaluation over real data is carried out in Section 4. In Section 5 we present the prototype built over the cTabSum algorithm and how the user can interact with the resulting summarized table. We also supply an illustrative example with the purpose to realize a qualitative evaluation of our approach. Section 6 concludes and describe some possible future works.

Related Work

A first approach proposed in the context of table summarization was presented in [?]. This approach creates and maintains table summaries through row and column reductions. To reduce the number of rows, the algorithm first partitions the original table into groups based on one or more attribute values of the table, and then collapses each group of rows into a single row relying on the available metadata, such as the concept hierarchy. In [?] the SaintEtiQ is proposed. This system computes and incrementally maintains a hierarchically arranged set of summaries of the input table. SaintEtiQ uses background knowledge (i.e., metadata) to support these summaries. [?] proposed to work at the metadata level to improve the final summarization and speed-up the whole process. The strategy is based on a pre-processing of the original concept taxonomy in order to obtain a more compact hierarchy. Once the reduced metadata is available it is employed to perform the table summarization task. As we can observe, all the previous algorithms exploit some metadata knowledge that compulsoliry need to be supplied by the user. On the contrary, our approach is able to perform and complete the process of table summarization without asking any additional information to the expert. This characteristic allows cTabSum being much more flexible and usable when no background knowledge is available.

cTabSum

In this section we first introduce preliminary notations and then describe in details the cTabSum framework. We define a table T as a set of tuples, i.e., T = {t 1 , t 2 , ..., t n }. Each t i is described over a set of attributes A 1 , . . . , A m , i.e., t i ∈ (dom(A 1 ) × dom(A 2 ) × ... × dom(A m )). Given 1 ≤ i ≤ n and 1 ≤ j ≤ m, T ij denotes the value of the attribute A j in the i th tuple of T . Given an attribute A j , V is a partition over the values of that attribute such that ∀ v∈V v ⊂ A j and ∀ v,w∈V v ∩ w = ∅. The partition V induces a generalization of the attribute A j . More precisely, given a value a jk ∈ dom(A j ) (a jk is the k-th value of the attribute A j ), it exists a value v s.t. a jk ∈ v. For instance, given an attribute taxonomy over A j , the ancestor of an attribute values can be seen as a generalization of it. The objective of the TSP is to find a summarized table s(T ) = {g(t 1 ), g(t 2 ), ..., g(t l )} s.t. each g(t b ) (generalized tuple) cover at least k tuples of the original table T and there are no overlap among the tuple set covered by generalized tuples.

Proposed Approach

Essentially the proposed strategy proceeds in two main steps. The first step consists in building the cAVTs (one for each attribute A j over which T is defined). Leveraging data mining techniques, we can extract this meta-information starting from the data [?]. The second step performs the table summarization strategy. The algorithm uses the previously calculated cAVTs to generalize the data. To do so, a new approach that exploits a criterion coming from information theory to evaluate the quality of the possible solutions in the search space is proposed. Similarly to [?], cTabSum also requires a parameter k but it avoids any metadata information (taxonomies) as input.

The TSP extracts a summarized table with the constraint that each generalized tuple must cover at least k tuples of the original table , where k is a user-defined parameter. The table summarization task can be related to the kanonymity problem [?]. While the algorithmic approaches could be similar, the final results are different in spirit. TSP supplies an informative compression of data to allow exploratory analysis in contrast to the k-anonymity purpose which aims at hiding some sensitive information. Another difference is that table summarization is performed for all the attribute of the data table while k-anonymization involves only the set of attribute composed by quasi-identifiers (sensible attributes).

Table-Summarization Quality Criterion

The TSP task can be seen as a search problem in which we want to find, if possible, the best compression of the original table taking into account the constraint k. This best compression also implies to have the minimum information loss w.r.t the original data. Thus, when facing with a set of possible intermediate solutions, it is needed to understand which one should be chosen. In the field of data compression, information theory measures are normally used to evaluate, given a model, the gain in compression (and information loss) w.r.t. the uncompressed data [?]. As our final goal is to obtain a summarization of the original table, these kind of measures can be very suitable. Particularly, to evaluate how much information is lost during the summarization process, we employ the nonuniform entropy [?]. The non-entropy measure, that we call Inf oLoss(T, s(T )), is defined as follows:

Inf oLoss(T, s(T )) = n i=1 m j=1 -log P r(T ij = a jk |s(T ) ij = v) (1)
where

P r(T ij = a jk |s(T ) ij = v) = #{1 ≤ i ≤ n : T ij = a jk } #{1 ≤ i ≤ n : T ij ∈ v}
The conditional probability P r(T ij = a jk |s(T ) ij = v) indicates how the subset of original values of A j are distributed w.r.t. their generalization induced by partition v. The non uniform entropy measure (w.r.t. the classical entropy measure) helps to better considering the distribution of the values belonging to the same generalization.

Building CAVTs

An important step of our strategy is the automatic construction of the cAVTs.

To structure attribute values in taxonomies we exploit the approach proposed in [?]. This technique allows to extract distances between each pair of values of the same categorical attribute. Most in detail, given a categorical attribute A j , over which the database D is defined, it selects a set of other attribute strictly related to it using correlation measure. This set of attribute is called context. After that, it uses the distribution of the values of attribute A j w.r.t. the attributes in the context to infer a distance matrix that represents the distances between each pair of values v jk , v jl ∈ A j , where v jk is the k-th value of attribute A j . Once the point-wise distance matrix for A j is available, it is used as input for standard agglomerative hierarchical clustering[?] that produces the final cAVTs[?]. This approach is very useful when the original hierarchies are not available or to compare the induced taxonomies with the original ones understanding which one fit the actual analysis. As the TSP process requires hierarchies to summarize the original data, the cAVTs always supply this metadata knowledge. In particular, given an attribute, any cuts of the corresponding cAVT results in a partition of the domain of that attribute. Each of these partitions represents a possible generalization that can be used in the summarization process.

Constructing the Summary

At this point we are able to extract attribute taxonomies directly from data (cAVTs) and evaluate how far is a summary w.r.t. the original data (InfoLoss). Now we need to design a strategy to navigate the space defined over all the possible solutions. In this direction we design an algorithm for TSP following the general idea presented in [?]. There are three big differences between the proposed method and the one described in [?]:

1 cTabSum supplies a summary of the original data for exploratory purpose while in [?] the final goal is the anonymization of the data following the k-anonymity philosophy [?]. 2 In [?] the goal is to preserve classification accuracy and for this reason the class variable is used to guide the privacy preservation process. cTabSum is designed for a totally unsupervised scenario where no class information is available and the final goal is to compress the original data. To implement this important difference, instead of the Information Gain used in the privacy preserving strategy we minimize the Information Loss criteria based on Non Uniform Entropy. 3 cTabSum does not require any metadata because it directly mines this knowledge from the data relaxing the strong assumption on the availability of such kind of information.

The general process is presented in Algorithm 1. It takes as input a value k and returns a set of generalized tuples that describes the original table. The parameter k indicates the minimum number of original tuples that need to be covered by each generalized tuple in the final result. The search strategy is performed by a top-down navigation over the space of possible solutions. The top-down search starts from the top of all the attribute value taxonomies (the coarsest granularity levels) and, at each step, tries to specialize and expand only one attribute taxonomy with the function bestExpansion(T, H, x). This function chooses to expand the cAVT that produces the minimum loss of information evaluated by the Formula 1. Most in detail, for each attribute A j the following steps are performed: (i) generate all the possible expansions starting from the actual frontier of the cAVT, (ii) choose the best expansion that minimize the measure in Formula 1. As the process is performed over all the attributes simultaneosuly, bestExpansion(T, H, x) can expand any of the taxonomies at any node. This means that in the final summarized table, for the same attribute, we can have a generalization (partition) at different levels of granularity. Once that the actual expansion y is chosen we evaluate both: the Information Loss w.r.t. the actual best solution and the constraint supplied by k. If both conditions are satisfied, y is marked as new best solution. At the end the algorithm returns the solution that minimize the Information Loss and, at the same time, the constraint k.

cT abSum is implemented in a tool that has the same name and it is public available at the url address: http://www.lirmm.fr/ ~ienco/Dino_Ienco_Home_ Page/cTabSum.html.

Experiments

In this section we draw some experiments to analyze the performance of our approach using real world data table. The approach has been implemented in Java Algorithm 1: cT abSum(T , k) and we carried out experiments on Mac OS X 10.6.8 with 4Gb of RAM and an i7 2.2GHz processor. Actually this task shares similarities to the k-anonymization problem, in which each tuple of the anonymized table needs to represent at least k tuples of the original table. For this reason we used the algorithm MinGen presented in [?] as competitor to evaluate the performance of cTabSum. This algorithm was originally developed for the k-anonymity problem. We couple Min-Gen with the original available taxonomies. The algorithm needs two parameters. The first parameter k is the minimum number of tuples indistinguishable over the set of sensitive attributes (quasi-identifiers) [?]. The quasi-identifiers are the attributes for which the generalization process is performed (and the attribute taxonomies is demanded). The second parameter is the suppression coefficient sc standing for the maximum number of tuples (in percentage w.r.t. the size of the table) that the algorithm suppresses to obtain a k-anonymized table. This parameter is not relevant for the table summarization task and it has thus been set to 0. To evaluate the performance we use the Adult dataset5 . It is based on census data and has been widely used to evaluate classification and k-anonymization algorithms. Here, the same settings as in [?] are used. We obtain a dataset of 8 attributes. The attribute age is discretized in 8 equal-size bins. Starting from this dataset we sample 10% of the original dataset obtaining 4 522 tuples. As original hierarchies we use the taxonomies supplied in [?]. In our experiment we range the k parameter from 40 to 400 at step of 40. To evaluate the quality of the compressed table we adopt the non-uniform entropy measure (Formula 1). The idea is that a good k-anonymization (for this reason also a good summary of an original table) is the one that minimizes the Information Loss respecting the given constraints. As cTabSum generates attribute taxonomies directly from the data, a satisfactory result could be to obtain comparable performances compared to the ones involving the original hierarchies. In Figure 1 we observe the behaviors of the different approaches. Generally, we can state that the two approaches have comparable performances. This is a good point for cTabSum that automatically generates attribute taxonomies from data. In particular for values of k lesser than 240 cTabSum obtains better results in terms of Information Loss. This value of 240 influences seriously the final summarization because for values above this threshold both algorithms return summaries of only 4 generalized tuples to represent the original data table. From an user point of view, this result can be useless due to the fact that it compresses to much the original information. With values lesser than 240, both approaches return more reasonable summarization composed at least of 8 tuples. These results can represent more reasonable starting points for an initial explorative analysis of the data.

Assessing Summarization via Swap Randomization

In this subsection we evaluate how far the results of cTabSum are from randomness. A table summarization could be seen as a partition of the original tuples in groups that share common characteristic w.r.t. the attribute taxonomies. In this way TSP produces a clustering of the tuples constrained by metadata. Unfortunately we do not have any background knowledge to evaluate the quality of this partition. For this reason following the idea proposed in [?], we compare our results w.r.t randomized partition. Practically, given a table summarization result, each generalized tuple represents a cluster of the original ones. Starting from this clustering result, we swap at random, tuples between clusters obtaining random partition. In the random partitions the marginal distribution given by the original clustering are maintained. To evaluate the clustering solution we employ the ZIP function always available over any OS. In particular we zipped each cluster separately and we sum the size of all of them for a clustering solution in order to obtain a value for each value of k. The value is expressed in Kb. Low values indicate high compression rate that means a kind of structure inside the partitions. We employ this kind of measure because because it naturally captures how good is the compression of the original table. The ZIP procedure also constitutes an easy way to evaluate this quantity. Figure 2 shows the results. For each value of k we generate 100 random clustering following the distribution induced by the result of cTabSum and we report the average value. We avoid to visualize the standard deviation because it is always smaller than 1kb. We can observe that the results produced by cTabSum always give a lower size of the final ZIP files, this underline how the clustering induced by cTabSum contains a structure that can be easily recognize by a compression algorithm as the one implemented by the ZIP function.

The cTabSum System

As we have obtained interesting results from an experimental point of view, our algorithm has been integrated in a prototype named cTabSum. cTabSum is a standalone software written in Java. It is based on several open source projects. First of all, it uses the Weka Data Mining Library to manage the data1 . More precisely, our system requires a file in ARFF format (Attribute Relational File Format). In the GUI we integrate the JFreeChart Library2 to produce and visualize 2D chart and the JUNG (Java Universal Network/Graph) library3 to visualize attribute taxonomies.

Figures 3(a is described over a set of discrete features (e.g., buying, safety, type, etc..). Once the TSP is launched, the final table summarization result is shown in the bottom part of the GUI. The result of the summarization process is displayed in table that contains all the original attributes plus an extra attribute, Count, to count how many original tuples are covered by each generalized tuple. The * symbol indicates that the root level of the corresponding attribute taxonomy is used, i.e., all the values are grouped together. In the proposed example, the algorithm has found a solution that involves the specialization of only two attributes: safety and type. Starting from this result the user can interact with the data. The combo box in the top left part of the window allows to choose and investigate a particular attribute of the dataset. In the middle of the window, the cAVT can be visualized. In the tree visualization panel, two different types of nodes coexist: triangle nodes represent group of two or more original values while circle nodes represent group of only one attribute value.

In this example, we observe that the taxonomy associated to the attribute Type is cut in a way to produce two groups of attributes: the left node with only one value, unacc (unacceptable), and the right node that contains a group of three values, acc, good and vgood. The actual taxonomy cut can be also visualized in the right bottom part of the interface in which each cluster is described with its representative plus the enumeration of the attribute values that it contains (clustering list). The user starts from this taxonomy cut to browse the hierarchy using drill-own and roll-up operators. These operations modify the displayed attribute taxonomy. The changes in the attribute taxonomy are propagated to all the other components in the GUI, i.e., Generalized table and Cluster list. In Figure3(b) the application displays the results after an user action over the attribute taxonomy. We can observe that the user has expanded the taxonomy Type thanks to a drill-down operation. It should be noted that this expansion impacts on both the panels Generalized table and Cluster list that now displays the current view of exploration. This means that the Generalized table panel visualizes the generalized tuples correspondingly to the actual cut of the different attribute taxonomies while the Cluster list shows the partition of leaves of the selected attribute with respect to the considered level of granularity.

Another feature implemented in cTabSum is shown in the right upper side. For each attribute, a 2D chart is plotted where the attribute values are positioned in a way that preserves the relationships existing in the original pointwise distance matrix. When a user selects a new attribute from the combo box, both the chart and the taxonomy are automatically updated to display information related to the newly selected attribute. As an additional feature supplied by our prototype, when there some attribute in the dataset are numerical, cTabSum exploits the pre-processing facility supplied by Weka and performs a discretization step deriving 10 equal-width bins for each numerical attribute. In this way our system is able to manage tables containing mixed attributes types. The cTabSum implementation is available at the URL: http: //www.lirmm.fr/ ~ienco/Dino_Ienco_Home_Page/cTabSum.html.

Conclusion

The process of Table Summarization helps the analyst to have a first picture of the data. In our work a new algorithm to perform table summarization cTabSum is proposed. One of the main features of cTabSum consists in relaxing the strong assumption on the availability of attribute taxonomies. This is realized mining directly the attribute taxonomies from the data. Associated with the new algorithm we also developed a java prototype designed to fully exploit the knowledge available in the data. It uses cTabSum to aggregate the original data to supply (i) a more compact representation of them (ii) a starting point from which the user can navigate the table. More precisely, starting from the result of our system, the user can browse, interact and modify the data summary

Fig. 1 .

 1 Fig. 1. Information Loss of cTabSum and M inGen over the Adult dataset

Fig. 2 .

 2 Fig. 2. Size in Kb of zip files comparing the group obtained by cTabSum and the randomization result

Fig. 3 .

 3 Fig. 3. The cTabSum system.

In this paper we will use these two terms indistinguishly.

http://archive.ics.uci.edu/ml/

http://www.cs.waikato.ac.nz/ml/weka/

http://www.jfree.org/jfreechart/

http://jung.sourceforge.net/

navigating through the attribute taxonomies performing both drill-down and roll-up operations. In the future we plan to integrate cTabSum in open source OLAP project (like Mondrian) in order to supply a sort of recommendation to the final user to start his/her analysis. From an algorithmic point of view we want eliminate the use of parameter k replacing it with a lower bound concerning the minimum number of generalized tuples the final result may contain. Finally, the goal of our study is to summarize simple relational table, in which static information appears. In a next step, summarizing more complex data, e.g., data with temporal dimension, could be considered. In such a case, the generation of cAVTs will be different and much complicated and require further investigations.


Behnaz Behmand

Evangelos Balanikas

Lara Martinez-Fernandez

Roberto Improta

A Banyasz

Gerard Baldacchino

Dimitra Markovitsi

Correction to "Potassium ions enhance guanine

Correction to "Potassium Ions Enhance Guanine Radical Generation upon Absorption of Low-Energy Photons by G-Quadruplexes and Modify Their Reactivity" Behnaz Behmand, Evangelos Balanikas, Lara Martinez-Fernandez,* Roberto Improta, Akos Banyasz, Geŕard Baldacchino, and Dimitra Markovitsi* J. Phys. Chem. Lett. 2020, 11, 4, 1305-1309. DOI: 10.1021/acs.jpclett.9b03667 Cite This: J. Phys. Chem. Lett. 2020Lett. , 11, 2742Lett. -2742 Read Online

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Metrics & More Article Recommendations F igures 2 and 3 have been inverted, and the figure captions, which are in the correct position, do not correspond. The correct correspondence is as follows. Addition/Correction pubs.acs.org/JPCL

Figure 2 .

 2 Figure 2. Quantification of hydrated electrons ejected from (TG4T) 4 /K + (1.5 × 10 -5 mol L -1 ) upon 266 nm excitation. (a) Transient absorption decay at 700 nm obtained with incident intensity of 1.7 × 10 6 W cm -2 . (b) Ionization curve; [hν] and [e hyd -] 0 denote, respectively, the concentration of absorbed photons per laser pulse and the zero-time concentration of hydrated ejected electrons. Red lines represent fits with model functions: A 0 + A 1 exp(-t/τ 1 ) (a) and [e hyd -] 0 /[hν] = ϕ 1 + α[hν] (b).

Figure 3 .

 3 Figure 3. Differential absorption spectra determined for (TG4T) 4 /K + at 3 μs (a; red), 100 μs (b; green), 2 ms (b; yellow), and 6 ms (b; blue). In (a) ΔA was divided by the initial concentration of hydrated ejected electrons [e hyd -] 0 ; the gray line in panel (a) shows a linear combination of the spectra corresponding to the radical cation (40%) 29 and the (G-H2) • radical of monomeric guanosine (60%), 30 considered with their ε values. ΔA in (b) was normalized to 1 at 585 nm.