Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
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High species turnover of the ant genus Solenopsis (Hymenoptera : Formicidae) along an
altitudinal gradient in the Ecuadorian Andes, indicated by a combined DNA sequencing
and morphological approach
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Thibaut DelsinneA,C, Gontran SonetB, Zoltán T. NagyB, Nina WautersA, Justine JacqueminA and
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Maurice LeponceA
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A
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Brussels, Belgium.
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B
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1000 Brussels, Belgium.
Biological Evaluation Section, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000
Joint Experimental Molecular Unit, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-
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C
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Solenopsis is a widespread ant genus and the identification of its species is notoriously difficult. Hence,
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investigation of their distribution along elevational gradients is challenging. Our aims were (1) to test the
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complementarity of the morphological and DNA barcoding approaches for Solenopsis species identification, and
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(2) to assess species diversity and distribution along an altitudinal gradient in the Ecuadorian Andes. Ants were
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collected in five localities between 1000 and 3000 m above sea level. In total, 24 morphospecies were identified
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along the gradient and 14 of them were barcoded. Seven morphospecies were confirmed by the molecular
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approach. Three others, occurring sympatrically and possessing clear diagnostic characters, showed low genetic
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divergence. Representatives of a further four morphospecies were split into nine clusters by COI and nuclear
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wingless genetic markers, suggesting the existence of cryptic species. Examination of gynes revealed potential
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diagnostic characters for morphological discrimination. Solenopsis species were found up to an altitudinal record
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of 3000 m. Most morphospecies (20 of 24) were found at a single elevation. Our results suggest a high species
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turnover along the gradient, and point to the use of morphological and DNA barcoding approaches as necessary
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for differentiating among Solenopsis species.
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IS12030
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High altitudinal species turnover of Solenopsis ants.
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T. Delsinne et al.
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Manuscript received 18 April 2012, accepted 16 September 2012, published online dd mmm yyyy
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Introduction
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Solenopsis Westwood, 1840 is a large myrmicine ant genus encompassing 183 species, with a
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worldwide distribution (Guénard et al. 2010; Bolton 2012). The most well known species of this genus
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inflict a painful sting and are known as fire ants. Some fire ants, such as Solenopsis invicta, have
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become important invasive pests (Tschinkel 2006). Other species are referred to as thief ants because
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some of them are known to steal food from other ants (Pacheco 2007). About half of all described
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Solenopsis species are found in the Neotropical region (Fernández and Sendoya 2004). Solenopsis
Corresponding author. Email: Thibaut.Delsinne@sciencesnaturelles.be
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nests can be found virtually everywhere, in soil, leaf litter, dead wood, epiphytes or plant cavities
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(Creighton 1950). Workers forage from deep in the soil (Ryder Wilkie et al. 2007) to high in the forest
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canopy (Blüthgen et al. 2000). They are one of the most frequently encountered ant genera in ground-
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dwelling ant communities (Ward 2000; Donoso and Ramón 2009; Braga et al. 2010) and, as a
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consequence of their diversity and abundance, they are considered to be of significant ecological
40
importance in the Neotropics (Ward 2000).
41
Members of the genus can be easily differentiated from other Myrmicinae by the 10-segmented
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antenna with a 2-jointed club, the propodeum rounded and unarmed, the petiole and postpetiole nodes
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well developed, and the clypeus longitudinally bicarinate with an isolated median seta (Ettershank
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1966; Bolton 2003). Identification to specific level is, however, extremely difficult. Mackay and
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Mackay (2002) indicated that ‘identification is nearly impossible’. Creighton (1930), in his incomplete
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revision of the New World Solenopsis, wrote: ‘Carlo Emery once characterised the genus Solenopsis
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as the crux myrmecologorum. That the term is apt no one who has experienced the difficulties of the
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group will deny, least of all the author who, at the end of three years of study, still finds the «cross» a
49
heavy burden’. In fact, the literature abounds with quotations describing similar opinions. For
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instance: ‘the genus Solenopsis is no favorite of ant taxonomists’ (Thompson 1989), ‘at least some of
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[Solenopsis] are exceedingly difficult to classify’ (Smith 1943), ‘the members of the thief ant group of
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the genus Solenopsis have had a notorious reputation of being difficult to identify for over 70 years …
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this reputation is merited’ (Pacheco 2007). Snelling (2001) indicated, while describing S. maboya, a
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new thief ant from Puerto Rico, that ‘maboya is the Taino (Arawak) word for a perverse spirit, and
55
seemed appropriate, given the challenging nature of the taxonomy of this group of ants’.
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The difficulty of identifying specimens of Solenopsis is explained by two factors. First, worker
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morphology monotonously lacks diagnostic characters. Thief ants are tiny, often less than 2 mm long,
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which complicates the recognition of morphological characters. Fire ants are larger but polymorphic,
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presenting a continuum of sizes in the same nest. For instance, workers of S. invicta range from 2.65
60
mm to 6.16 mm in body size (Tschinkel et al. 2003). Moreover, all species of fire ants and several
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thief ant species exhibit intraspecific variation in morphological traits which may exceed interspecific
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differences (Pitts et al. 2005; Pacheco 2007; Ross et al. 2010). Males and gynes may be less uniform
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morphologically and offer additional characters for species identification (Creighton 1950). However,
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these reproductive castes are less frequently encountered and rarely associated with workers
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(Creighton 1950; Pacheco 2007). Second, most species were inadequately described on the basis of
66
limited material, mainly between the end of the 19th and beginning of the 20th centuries. The use of
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numerous trinomials and quadrinomials has generated serious taxonomical confusion (Pacheco 2007).
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Creighton (1930) attempted to revise New World Solenopsis but most thief ants were not included in
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his work since he planned to treat them in a separate publication which never eventuated. Trager
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(1991) restricted his revision to fire ants but, even afterwards, species delimitation often remains
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problematic (Ross et al. 2010). More recently, Pacheco (2007) revised New World thief ants, proposed
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eight species complexes and numerous new synonymies and other changes, recognised 83 species and
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presented keys for the identification of workers. Unfortunately, this thesis does not conform to the
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rules of the International Code of Zoological Nomenclature, making it impossible to acknowledge his
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taxonomical changes (William Mackay, pers. comm.).
76
Thus, the α-taxonomy of Solenopsis is still confused, which represents a serious impediment for ant
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biodiversity inventories, and ecological work in general. Most collected species are misidentified
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(Thompson 1989) or are simply recorded only as morphotypes. For instance, 13 Solenopsis species
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were sampled in the Nouragues Research Station, in French Guiana, but only two of them could be
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assigned to a valid name (Groc et al. 2009). Similarly, only one of 15 species collected during a
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thorough inventory in Ecuadorian Amazonian forests was named (Ryder Wilkie et al. 2010).
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Recently, the use of DNA barcodes, short mitochondrial DNA sequences of the cytochrome
83
oxidase I (COI) gene, has been proposed to facilitate species identification and discovery (Hebert et al.
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2003; Janzen et al. 2009). This method is acknowledged as a useful explorative tool to provide
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estimates of species numbers, especially in very diverse and poorly understood taxonomic groups
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(Wiemers and Fiedler 2007; Jansen et al. 2009; Strutzenberger et al. 2011; Tänzler et al. 2012). In
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particular, DNA barcoding has proved useful in complementing morphological species determination
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in biodiversity surveys of ants (Smith et al. 2005; Fisher et al. 2008; Fisher and Smith 2009), in
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facilitating caste association (Fisher et al. 2008), and assisting in the discovery of new cryptic ant
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species (Schlick-Steiner et al. 2006; Fisher et al. 2008; Menke et al. 2010). Nevertheless, the
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barcoding approach possesses several pitfalls and shortcomings (reviewed in Rubinoff et al. 2006;
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Jinbo et al. 2011) with barcoding success rate varying among taxa (Elias et al. 2007; Jansen et al.
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2009; Wild 2009). Therefore, species hypotheses based on DNA barcodes should be supported by
94
additional, independent nuclear markers (Ross et al. 2010; Smith et al. 2011) or other data such as
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morphology, geography, ecology or behaviour (Yassin et al. 2010).
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For unknown reasons, a previous attempt to amplify the COI marker from thief ants was not
97
successful (Pacheco 2007) and, so far, most genetic studies of Solenopsis have focussed on fire ants
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(Ross and Shoemaker 2005; Shoemaker et al. 2006; Ross et al. 2010). These analyses have identified
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genetically independent lineages within variable and widespread taxa. Further, the combined use of
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mitochondrial and nuclear markers have revealed cryptic species (Ross et al. 2010).
Identification of Solenopsis species is expected to be particularly challenging along elevational
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gradients, where it is frequently found that ants once considered to belong to a single widely
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distributed species turned out to be several cryptic species with parapatric distributions and restricted
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altitudinal ranges (Lattke 2003). Our aims in this study were (1) to test the complementarity of
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morphological and DNA barcoding approaches for species identification in the genus Solenopsis, and
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(2) to assess species diversity and distribution along an altitudinal gradient in the Ecuadorian Andes,
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which is considered to be a biodiversity hotspot for many taxa.
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Materials and methods
Ant sampling
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Ants were collected between 2007 and 2011 in seven forested sites spread among five localities
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between 1000 m and 3000 m above sea level with elevational steps of 500 m between localities. Study
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sites were selected in the Podocarpus National Park and two adjacent protected areas (Reserva
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Biológica San Francisco and Copalinga private reserve), on the eastern range of the South Ecuadorian
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Andes, in the provinces of Loja and Zamora-Chinchipe. Details about the study area are provided in
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Beck et al. (2008). Five reference sites were selected: Copalinga Private Reserve –blue trail (called
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hereafter ‘1050 m-C’; 4°5S, 78°57W), Copalinga Private Reserve – red trail (‘1420 m’; 4°5S,
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78°58W); Reserva Biológica San Francisco – Transect T1 (‘2070 m-R1’; 3°58S, 79°5W), El Tiro
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(‘2500 m’; 3°59S, 79°7W) and Cajanuma-Podocarpus National Park (‘3000 m’; 4°6S, 79°10W).
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Two supplementary sites were sampled at 1050 and 2070 m: Bombuscaro-Podocarpus National Park
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(‘1050 m-B’; 4°6S, 78°58W) and Reserva Biológica San Francisco-NUMEX (‘2070 m-R2’; 3°58S,
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79°4W). The distance between sites ranged from 2 to 20 km. At each site, ants present in quadrats of
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leaf litter were extracted by the Winkler method (54 m2 extracted per site). In addition, we searched
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for Solenopsis nests in dead wood, soil and vegetation in an attempt to document reproductive castes
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in association with series of workers. Specimens were preserved in 96% ethanol (denatured with
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diethyl ether) and sorted to morphospecies on the basis of criteria proposed by Pacheco (2007), such
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as expression of clypeal teeth, number of ommatidia, number of mandibular teeth, scape length, body
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colour, pattern and extent of sculpture, shape and size of body tagma, expression of anteroventral
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petiolar process and size of cephalic punctures. We used the phenetic species concept and expected
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that a certain degree of difference in morphological characters indicated potential reproductive
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isolation.
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A few specimens from each morphospecies were pinned and photographed. Images were taken with
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a Leica DFC290 camera attached to a Leica Z6APO stereomicroscope. A series of images was taken
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by focusing on different levels of the insect, using the Leica Application Suite v38 (2003–2011) and
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combined with CombineZP (Hadley 2010). Final processing of images was done in Adobe Photoshop
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CS5. Original images were deposited in Morphbank (collection no.: 801203;
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http://www.morphbank.net/801203). Voucher specimens were deposited at the Royal Belgian Institute
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of Natural Sciences, Brussels, Belgium, and at the Universidad Técnica Particular de Loja, Loja,
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Ecuador.
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Laboratory method
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About 10 260 Solenopsis specimens were collected during this study (up to 4605 specimens per site).
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Multiple representatives (n = 2–70) of each of the most abundant Solenopsis morphospecies were
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selected for DNA analysis. Seven morphospecies represented by fewer than five individuals were
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discarded. Analyses were carried out on 187 Solenopsis specimens (Supplementary material S1). Total
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genomic DNA was isolated from the complete ant body using the commercial NucleoSpin Tissue Kit
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(Macherey-Nagel, Germany). After DNA extraction, specimens were preserved as vouchers in
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absolute ethanol. Amplification of the mitochondrial cytochrome c oxidase subunit I (COI) marker
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was carried out in polymerase chain reaction (PCR) using the primer pair LF1 and LR1 (Smith et al.
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2005) modified from Hebert et al. (2004a) and the universal primers LCO1490 and HCO2198 (Folmer
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et al. 1994). When amplification systematically failed, DNA quality was checked on 1.2% agarose gel,
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and smaller DNA fragments were amplified using the primer combination LCO1490 and LCO-
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ANTMR1D-RonIIdeg_R (Fisher and Smith 2008) modified from Simon et al. (1994). Amplification
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of the nuclear wingless (wg) marker was performed for a selection of 1–3 sample(s) per COI
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haplogroup using primers wg578F (Ward and Downie 2005) and wg1032R (Abouheif and Wray
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2002). Each PCR was prepared in a total volume of 25 µL containing 2 µL of DNA template and 0.03
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U µL–1 of Platinum® Taq DNA polymerase (Life Technologies, USA), 1 PCR buffer, 0.2 mM dNTPs,
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0.4 μM of each primer, 1.5 mM MgCl2. PCR protocol followed the profile of 94°C for 3 min; 5 cycles
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of 94°C for 30 s, 45°C for 30 s and 72°C for 60 s; 36 cycles of 94°C for 30 s, 50°C for 30 s and 72°C
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for 60 s; followed by a terminal elongation step at 72°C for 7 min, and subsequent storage at 4°C. PCR
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products were visualised on ~1.2% agarose gel, and purified using the NucleoFast 96 PCR Plate
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(Macherey-Nagel, Germany). PCR products were sequenced with an ABI 3130xl automated capillary
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sequencer using BigDye v1.1 chemistry and following the manufacturer’s instructions (Life
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Technologies, USA).
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Genetic data analysis
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DNA sequences were checked for quality and aligned by hand. No internal stop codons were detected.
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Homologous fragments of COI sequences of Solenopsis available in GenBank and BOLD
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(Ratnasingham and Hebert 2007) were added to the dataset provided that no characters were missing.
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As the length of the sequences obtained varied from 237 to 658 bp, three datasets were created: one
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including a maximum number of samples but with short sequences (237 bp) and two with longer
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sequences but including fewer samples (310 and 631 bp). Distributions of pairwise uncorrected
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distances were plotted for all genetic datasets using the R language and environment for statistical
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computing and graphics ver. 2.14.2 (R developmental core team) and package ape v2.7-3 (Paradis et
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al. 2004). For an overview of the pairwise genetic distances, a neighbour-joining tree with
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bootstrapping (1000 replicates) was constructed on the basis of the uncorrected distance matrix of the
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631-bp dataset and using MEGA v5.01 (Tamura et al. 2011). Putative species delimitation was
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performed using uncorrected distances without a phylogenetic tree reconstruction in which the
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assumption of monophyly would be doubtful based on a single gene and incomplete sampling (Taylor
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and Harris 2012). In the absence of species identifications, intraspecific distances could not be
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distinguished from interspecific distances and no optimal threshold distance could be defined for
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species delimitation. For this reason, different threshold values were used. Since intraspecific
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distances are expected to be generally lower than interspecific distances – forming a ‘barcoding gap’ –
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(Hebert et al. 2004b), local minima in the distribution of genetic distances can be used as tentative
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threshold distance to test for delineation of species. All local minima of the density of the pairwise
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distances were determined using the function localMinima of package spider v1.1-2 (Brown et al.
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2012) and were used as thresholds. On the basis of the literature, we also selected threshold values of
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2% and 10%. The former was proposed as a standard distance for ants (Smith et al. 2005; Smith and
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Fisher 2009) and the latter represented an extreme value rarely surpassed by intraspecific distances
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(e.g. Smith and Fisher 2009; Yassin et al. 2010).
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Clustering of samples was performed for each threshold and based on pairwise distances using the
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function tclust of the package spider v1.1-2 (Brown et al. 2012). Samples showing genetic distances
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greater than the threshold with any member of a cluster were excluded from that cluster. For the
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cluster encompassing more than five different haplotypes, a haplotype network was calculated with
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pegas v0.4-1 (Paradis 2010) based on the longest fragment available for this subset of samples (658
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bp). Finally, a tree was calculated with the Bayesian method of phylogenetic inference, and based on
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the available concatenated sequences of COI (658 bp) and wg (343 bp).
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The dataset was partitioned into six, each partition representing separated codon positions. The
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Bayesian information criterion implemented in jModeltest v0.1.1 (Posada 2008) was used to find
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appropriate nucleotide substitution models for all partitions, and recommended settings were used in
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the subsequent Bayesian analysis. Analysis was carried out with MrBayes v3.1.2 (Ronquist and
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Huelsenbeck 2003) running 10 million generations in two runs. Each run involved four Monte Carlo
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Markov chains, one of them being cold and the three others heated using MrBayes’ default settings.
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Every 1000th generation was sampled. Split frequencies were observed, and the convergence of the
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chains was monitored by Tracer v1.5 (Rambaut and Drummond 2009). At the end of the run, the
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potential scale reduction factor was checked for all parameters, and was found to be close to 1.0. The
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first 25% of the sampled trees were discarded (‘burn in’) and the remaining trees were used to
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construct a consensus. COI and wg sequences were deposited in BOLD (Ratnasingham and Hebert
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2007) with Process ID from SOLEN001–12 to SOLEN110–12.
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Faunal similarity among sites
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The faunal similarity of Solenopsis species among sites was compared using the Jaccard index of
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similarity (J): J = A/(A+B+C), where for any Sites 1 and 2, A is the number of species present in both
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sites, B the number of species only at Site 1 and C the number of species only at Site 2. Hence, when J
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= 1, both sites host the same Solenopsis species, and when J = 0, Solenopsis species collected in the
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two sites are completely different. We restricted this analysis to the dataset containing the longest (631
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bp) sequences. Only specimens documented by both morphological and DNA barcode data were
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included. We compared matrices of similarity obtained with the morphological and DNA barcoding
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approaches by Mantel tests (24 iterations) using Mantel 2.0 (Liedloff 1999).
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Results
Species delimitation and DNA barcoding
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Overall, 24 morphospecies of Solenopsis were identified in our sampling in the Ecuadorian Andes.
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Seven of these morphospecies were rare (1–5 individuals) and not used for genetic analyses.
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Microscopic examination of voucher specimens after DNA extraction confirmed that most anatomical
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features useful for species determination were preserved (see extracted specimens on Figs S8, S17-S19
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in Supplementary material S2). COI sequences were obtained for 106 specimens representing 14
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morphospecies (no sequence was obtained for three of the morphospecies selected). Their lengths
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varied from 198 to 658 bp. Among them, 19 shorter sequences were obtained with the alternative
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primer combination: LCO-ANTMR1D-RonIIdeg_R. Datasets, including GenBank and BOLD
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sequences, consisted of (1) 245 sequences of 237 bp, representing 54 haplotypes, (2) 245 sequences of
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310 bp, representing 55 haplotypes, and (3) 213 sequences of 631 bp, representing 54 haplotypes.
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The DNA barcoding approach allowed us to successfully associate seven gynes and one male to the
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worker caste (identical sequences) for seven of the clusters separated by minimum 5%. In one cluster
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(Solenopsis sp. 16), worker, male and gyne castes were documented.
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The neighbour-joining tree based on the COI sequences of 631 bp (Fig. 1) showed genetic distance
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between haplotypes (from 0 to 21.4%). Local minima in the distributions of pairwise distances (p
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distance) were between 1.2 and 10% depending on the dataset (Fig. 2). Using these different
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thresholds, 21–36 clusters of similar sequences were defined for the entire dataset and, excluding
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GenBank and BOLD sequences, 14–20 clusters were defined for Solenopsis collected in Ecuador
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(Table 1). Sequences of the nuclear wg gene fragment (343 bp) were obtained for 26 Solenopsis
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workers. They represented 12 haplotypes and 11–12 clusters defined with COI using 5–1.2% distance
238
threshold, respectively. Genetic divergences among these nuclear sequences varied from 0 to 7% (Fig.
239
1, small tree). Before delimiting species based on COI sequences, we verified that no conflicts were
240
observed between the wg and the COI trees and detected no sign of hybridisation or other horizontal
241
gene transfer. Bayesian analysis of the concatenated COI–wg dataset resolved most of the nodes for
242
10–11 Solenopsis clusters (defined with COI using 5–1.2% distance treshold: Fig. 3). Three
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morphospecies (Solenopsis spp. 01, 14 and 15) that were morphologically similar were not sister
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species.
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All sequences obtained for COI from Ecuadorian Solenopsis specimens diverged considerably from
246
sequences available in GenBank and BOLD (p distance >9.2%). Five situations were observed:
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(a) Seven clusters were consistently discriminated using any threshold value and perfectly matched
248
249
morphospecies classification (‘Perfect match’ in Table 1).
(b) Seven other clusters were also consistently discriminated using any threshold value but
250
corresponded to four morphospecies (‘Splitting’ in Table 1). A thorough reexamination of
251
specimens recognised reliable diagnostic criteria for two of these genetically well defined clusters
252
(previously identified as Solenopsis sp. 01 [Fig. S12 in Supplementary material S2] and Solenopsis
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spp. 14–21 [Fig. S15 in Supplementary material S2]). The last five clusters corresponded to ants
254
presenting variation in their morphology but for which no reliable diagnostic criteria could be
255
identified (Supplementary material S2).
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(c) Three morphospecies presented clearly distinctive and consistent morphology and corresponded to
257
three genetically closely related groups (2%) of ants collected in the same locality at 2070 m
258
(‘Complex I’ in Table 1). The first one (Solenopsis sp. 11) included brown ants whose head,
259
mesosoma, petiole and postpetiole were uniformly foveate (Fig. S8 in Supplementary material
260
S2). The second one (Solenopsis sp. 12) corresponded to yellow ants with smooth head and
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pronotum and foveate mesonotum, propodeum, petiole and postpetiole (Fig. S10 in
262
Supplementary material S2). Ants from the third morphospecies (Solenopsis sp. 13) were entirely
263
smooth and pale yellow (Fig. S9 in Supplementary material S2).
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(d) In contrast, a deep genetic divergence (>10%) was found among workers of morphospecies 15
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(Table 1, Fig. 4A, B). A close examination of gyne morphology – associated with workers by
266
barcoding – supported the hypothesis that Solenopsis sp. 15 contains at least two cryptic species
267
(Fig. 4C–F).
268
(e) Lastly, specimens identified as Solenopsis spp. 01, 14 and 15 presented variable morphology (e.g.
269
body size, colour) and were genetically grouped together (‘Complex II’ in Table 1). In this case,
270
clustering based on DNA and morphology were not consistent. The haplotype network of 28
271
sequences of 658 bp showed that the observed genetic variation was related to elevation (Fig. 5).
272
No haplotypes were shared between specimens found at different altitudes or at the same altitude
273
but different sites. Furthermore, haplotypes found at lower (1050 m) or higher (2070 m) altitudes
274
were always connected with haplotypes found at intermediate elevation (1420 m).
275
Diversity and distribution of Solenopsis
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Both morphological and DNA barcoding approaches suggested a clear disparity among sites in terms
277
of species composition (Table 1). On the basis of morphology, only two morphospecies (Solenopsis
278
spp. 01 and 15) were found at 1050, 1420 and 2070 m, and two (Solenopsis spp. 07 and 16) at 1050
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and 1420 m. Solenopsis spp. 01 and 15 belonged to the same complex (‘Complex II’ in Table 1),
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which possessed genetic variation related to elevation (Fig. 5). The presence of Solenopsis sp. 16 at
281
‘1050 m-B’ could not be confirmed by DNA barcoding because no DNA sequences were obtained
282
from specimens of the corresponding morphospecies from this elevation. Except for these four taxa,
283
all morphospecies and well discriminated genetic lineage (p distance >5%) were restricted to a single
284
altitude (Table 1). As a result, similarity of Solenopsis assemblages among sites was low. For instance,
285
with the morphological approach, the Jaccard index of similarity (J) ranged from 0 to 0.4, and with the
286
DNA barcoding approach, from 0 to 0.38 (using a 5% threshold and the 631 bp dataset) (Table 2).
287
Patterns of assemblage similarity among sites obtained with morphology and DNA barcoding were
288
similar, and not dependant on the threshold (Table 2) (Mantel tests, 24 iterations, 0.85  r  0.90, P <
289
0.01).
290
Similarly, there was no significant difference between patterns of richness (defined as the number of
291
morphospecies or as the number of clusters obtained with the 631-dp dataset at a 10%, 5%, 2% and
292
1.2% threshold) using either the morphological or DNA barcoding approach (one-way ANOVA, F =
293
0.219, d.f. = 24, P = 0.925) (Fig. 6). Solenopsis richness was highest at mid-elevation (1420 m: Fig.
294
6). No specimens were collected at 2500 m but this could be an effect of local conditions since only a
295
handful of other ant species were found at this site. Two Solenopsis morphospecies, both represented
296
by a single worker (not included in the DNA analysis), were collected at 3000 m.
297
298
Discussion
Identification of Solenopsis using an integrative approach
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By combining morphological and genetic analyses, we were able to group Solenopsis specimens and
300
define units of biodiversity. Indeed, using different threshold distances to cluster DNA barcodes and
301
delineate potential species, we were able to distinguish between well delimited clusters that were
302
consistently grouped together and complexes of sequences showing gradual divergences. It is now
303
recognised that no single distance threshold can be universally applied in species identification
304
(Yassin et al. 2010). Nevertheless, the use of COI divergence among clusters may provide a
305
preliminary indication of species richness and help to detect unexpected cryptic species (Wiemers and
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Fiedler 2007; Burns et al. 2008; Tänzler et al. 2012). In ants, average interspecific sequence
307
divergences in COI generally exceed 2% (Smith et al. 2009; Wild 2009) although divergence within
308
and among species is not consistent (Wild 2009; Jansen et al. 2009). For instance, in Linepithema,
309
genetic divergences among species range from 0.5% to 7.8% (mean: 5.5%) while distances within
310
species ranged from 0% to 4.6% (mean: 1.9%) (Wild 2009). It is therefore necessary to use thresholds
311
with caution and to discuss cases of conflicts between morphology and barcoding results.
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In this study, 50% of Solenopsis morphospecies (7 of 14) were well defined by clear morphological
313
characters and were also separated from each other by relatively deep genetic divergence (5%;
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‘Perfect match’ in Table 1). Contrary to this, three sympatric morphospecies (Solenopsis spp. 11, 12
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and 13 – ‘Complex I’ in Table 1), easily separated on the basis of consistent morphological traits, but
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showed low genetic divergence (2%). For this complex, we hypothesise that three distinct species
317
were present and that distances among them were low perhaps because of recent speciation events. It
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has been shown previously that closely related species can differ by only 1–3 nucleotides (Burns et al.
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2007).
320
Four other morphospecies (Solenopsis spp. 01, 14, 15 and 21) were split into 9–15 clusters
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according to the available data and threshold considered (Table 1), suggesting that (1) morphological
322
criteria used in this study for species recognition were too conservative (see also Wild 2009), (2) some
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genetically distinct species could not be distinguished morphologically, or (3) some species presented
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intraspecific divergences over 10%.
325
Nuclear DNA analysis rejected the third hypothesis and supported Hypotheses 1 and 2 since
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morphologically similar specimens were not grouped together in a monophyletic clade. Such a result
327
has also been found in the hesperiid butterfly genus Perichares (Burns et al. 2008). Moreover, the
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DNA barcoding approach used here helped to detect cryptic species. For instance, some workers of
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Solenopsis sp. 15 were highly distinctive genetically (10%) but were very difficult to accurately
330
distinguish on the basis of morphology (Fig. 4A, B). A reexamination of the specimens did find
331
differences in shape of propodeum and of anteroventral petiolar process (Figs S11, S21 in
332
Supplementary material S2) but they were subtle and easily ascribed to intraspecific variations.
333
Fortunately, it was possible to associate gynes to workers of both clusters thanks to the DNA
334
barcoding approach (Fig. 4C, D). It seems that gynes provide more reliable criteria than workers for
335
separation of the two clusters (Fig. 4E, F). However, caution is needed because only 1–3 gynes were
336
associated with confidence to each cluster, making impossible the evaluation of intraspecific
337
variations of gyne morphology.
338
We admit that morphospecies sorting was probably too conservative in some cases. The fact that the
339
number of morphospecies was almost always lower than the number of clusters based on any
340
threshold of COI divergence (Fig. 6) suggests that lumping occurred during morphospecies
341
identification. The reexamination of specimens allowed us to find diagnostic characters to identify two
342
genetically well defined clusters (Figs S12, S15 in Supplementary material S2). Nevertheless, even
343
with the help the barcoding data, it was not always straightforward to distinguish intra- from
344
interspecific variation. For instance, specimens forming ‘Complex II’ (Table 1) were lumped together
345
using a 10% threshold but were separated into 3–5 clusters when lower thresholds were used. This
346
complex presented some genetic similarities (~10%) with specimens identified as S. molesta and
347
collected from Canada and the USA (Table 1; sequences obtained from BOLD). The molesta species
348
group sensu Creighton (1930) and Pacheco (2007) is a diverse, largely distributed complex of
349
morphologically very similar Solenopsis species. It is possible that this complex represents a
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monophyletic clade but supplementary studies are needed to confirm this hypothesis. Here, Complex
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II corresponded to morphologically variable specimens but no clear-cut criteria were found to separate
352
them. In addition, genetic divergence was related to elevation (Fig. 5). In the absence of evidence to
353
the contrary so far, we consider the variation observed within Complex II to be intraspecific variability
354
of isolated, diverging populations.
355
Patterns of diversity and distribution in Solenopsis
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Species richness of most ant genera decreases with elevation (Lattke 2003; Dunn et al. 2010). Here,
357
Solenopsis seems to be more abundant at mid-elevation but it is perhaps the consequence of
358
unsuccessful DNA extractions of specimens collected at 1050 m (Supplementary material S1). On the
359
basis of the integration of the barcoding and morphological data, numbers of Solenopsis species
360
collected were 4, 11 and 4 species at 1050 m-C, 1420 m and 2070 m-R1, respectively. Adding non-
361
barcoded, rare morphospecies and common species for which no barcode sequences were obtained
362
(hypothesising that morphology alone allowed correct identification in these cases), species richness at
363
reference sites reached 9, 13, 5, 0 and 2 species at 1050 m-C, 1420 m, 2070 m-R1, 2500 m and 3000
364
m, respectively. To our knowledge, records at 3000 m are the highest documented cases for the
365
occurrence of the genus. In total, we estimated that at least 30 Solenopsis species were collected along
366
the altitudinal gradient. Besides, similarity of Solenopsis assemblages among elevations was very low
367
(Table 2) and most Solenopsis species (25 of 30) were found at a single altitude, indicating that species
368
turnover and regional diversity were high. This is confirmed by the fact that sites at the same elevation
369
and less than 4 km apart shared only a few Solenopsis species (Tables 1 and 2).
370
It is difficult to compare our results with published data because sampling methods and effort were
371
different. Nevertheless, it is interesting to note that the local (α) diversity of Solenopsis found at 1050
372
and 1420 m are among the highest recorded. For instance, in the Otongachi forest (Ecuadorian Andes,
373
850 m), seven Solenopsis (morpho)species were collected with 40 pitfall traps and 40 1-m2 Winkler
374
samples (Donoso and Ramón 2009). In Tiputini Biodiversity Station, Amazonian Ecuador (206–224
375
m), 15 Solenopsis (morpho)species were inventoried from deep soil layers to canopy using six
376
sampling methods resulting in more than 200 samples (Ryder Wilkie et al. 2010). In an Amazonian
377
forest (Brazil, 30–140 m), 900 samples were collected with three sampling methods (1-m2 Winkler
378
samples, pitfall traps, and sardine baits: Oliveira et al. 2009), resulting in 15 Solenopsis
379
(morpho)species. Our results suggest that the Ecuadorian Andes are a hotspot of diversity for
380
Solenopsis ants and/or that the joint use of morphology and DNA barcoding allows better estimates of
381
Solenopsis local diversity than morphology alone.
382
Inventory of Ecuadorian Solenopsis species is still at an early stage. Fernández and Sendoya (2004)
383
found only two species, S. globularia and S. saevissima, cited from Ecuador in the literature. More
384
recently, S. virulens (Ryder Wilkie et al. 2010) and S. cf. stricta (Donoso and Ramón 2009) were
385
collected from the Amazonian region and the central Andes, respectively. Also, the invasive ant S.
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geminata was introduced to the Galápagos Archipelago and Ecuadorian mainland (Herrera and
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Causton 2008; Wetterer 2011), while S. gnoma, a suspected endemic of the Galápagos Islands, was
388
recently described (Pacheco et al. 2007). It is therefore clear that most of the Solenopsis species
389
collected in this study are potential new records for Ecuador and/or new species. Considering that 30
390
species is a good estimation of the Solenopsis diversity in our small study area (maximum distance
391
between sites was 20 km), we may expect that the estimated richness at a continental scale (i.e.
392
currently ~100 species are known in the Neotropic) is largely underestimated.
393
Despite the abundance and ecological importance of Solenopsis species (Ward 2000), these ants
394
remain poorly studied because of their problematic identification. This is a major impediment for
395
biodiversity and biogeographical studies. Our results show that morphological and DNA barcoding
396
approaches revealed similar patterns of species richness within sites and of species turnover among
397
sites, as observed for other ants in Madagascar (Smith et al. 2005). Montane rainforests in southern
398
Ecuador are recognised as biodiversity hotspots for numerous taxa (Brehm et al. 2008; Strutzenberger
399
et al. 2011). Given the high levels of species turnover among sites and of local and regional species
400
richness of Solenopsis it seems likely that this is also the case for ants. The combined use of
401
morphological and barcoding approaches increased the accuracy of Solenopsis identification. DNA
402
barcoding was also helpful to associate sexual and worker castes, adding potential new diagnostic
403
characters for species identification. In this respect, back and forth interactions between morphological
404
and DNA barcoding approaches were facilitated by the non-invasive DNA extraction protocol.
405
Acknowledgements
406
The authors warmly thank C. Vits and B. de Roover at Copalinga Private Reserve for access to their property, J.
407
Bendix, F. Matt, J. Zeilinger, the ‘Deutsche Forschungsgemeinschaft’ (DFG)-Research Unit 816, and the team of
408
the ‘Estación Científica San Francisco’ for allowing and extensively facilitating their work, I. Bachy, J. Cillis,
409
and Y. Laurent for ant digitisation, T.M. Arias-Penna and J. Peña for assistance during fieldwork. We also thank
410
A. Austin and two anonymous referees for comments and suggestions that greatly improved the manuscript. This
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research was funded by the Belgian Federal Science Policy Office (BELSPO) through an Action 1 Impulse for
412
Research and the Joint Experimental Molecular Unit (JEMU), and by the European Distributed Institute of
413
Taxonomy (EDIT). All material has been collected under appropriate collection permits and approved ethics
414
guidelines.
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604
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605
Table 1. Delimitation of Solenopsis species by morphological and DNA barcoding approach
606
Fourteen Ecuadorian morphospecies, for which COI sequences were available, are represented along
607
with Solenopsis records from GenBank and BOLD. Comparisons were made for three datasets: one
608
with short sequences (237 bp), and two with longer sequences but including fewer samples (310 and
609
631 bp). Threshold values corresponded to local minima in the distributions of pairwise distances and
610
to preset values (2% and 10%). Letters ‘a’–‘w’ refer to clusters of Ecuadorian Solenopsis. Letters
611
‘Ga’–‘Gp’ refer to clusters including sequences obtained from GenBank or BOLD. As a result, for
612
each threshold and dataset, the number of different letters in the column corresponds to the number of
613
clusters obtained with this threshold and dataset. Black cells mean that no sequence of the
614
corresponding length was obtained. Information on elevation and collection site is given. Brief
615
description of morphospecies and photos are provided in the Supplementary material S2 (Figs S1–
616
S24)
Potential
splitting
(Complex
II)
Splitting
Potential
lumping
(Comple
x I)
Perfect match
Morphospecies
2 (Fig. S1)
6 (Fig. S2)
7 (Fig. S3)
16 (Fig. S4)
18 (Fig. S5)
22 (Fig. S6)
19 (Fig. S7)
11 (Fig. S8)
13 (Fig. S9)
12 (Fig. S10)
15 (Figs S11, 4A, 4C, 4E)
1 (Fig. S12)
1 (Fig. S13)
14 (Fig. S14)
14,21 (Fig. S15)
1,15 (Fig. S16)
1 (Fig. S17)
1 (Fig. S18)
1 (Fig. S19)
1,14,15 (Fig. S20)
15 (Figs S21, 4B, 4D, 4F)
1,15 (Fig. S22)
10.0%
a
b
c
d
e
631 bp
5.0% 2.0%
a
a
b
b
c
c
d
d
e
e
h
h
h
k
l
m
n
o
p
q
h
h
h
k
l
m
n
o
p
q
h
i
j
k
l
m
n
o
p
q
s
s
s
s
s
t
u
u
s
t
u
u
Clustering based on COI
Altitude (m) and Site
310 bp
237 bp
1050 1420
2070
1.2% 10.0% 9.2% 5% 3.2% 2.0% 7,0% 3,1%
a
a
a
a
a
a
a
a
B,C
b
b
b
b
b
b
b
b
C
c
c
c
c
c
c
c
c
B
C
d
d
d
d
d
d
d
d
B?
C
e
e
e
e
e
e
e
e
C
f
f
f
f
f
f
f
C
g
g
C
h
h
h
h
h
h
h
h
R1,R2
i
h
h
h
h
h
h
h
R1
j
h
h
h
h
j
h
h
R1
k
k
k
k
k
k
k
k
C
l
l
l
l
l
l
l
l
R1
m
m
m
m
m
m
m
m
B
n
n
n
n
n
n
n
n
C
o
o
o
o
o
o
o
o
C
p
p
p
p
p
p
p
p
B
C
q
q
q
q
q
q
q
q
B
C
q
r
r
r
r
r
r
C
s
s
s
s
s
s
s
s
B,C
t
s
s
t
t
t
s
t
C
u
s
s
t
t
u
s
u
C
v
s
s
t
t
v
s
u
B,C
Page 18 of 26
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
15 (Fig. S23)
14 (Fig. S24)
Sequences from GenBank and BOLD
molesta
geminata
mameti
carolinensis
inv/richt
invicta
NZsp1
MU01
No. of clusters for Ecuadorian Solenopsis
Total no. of clusters
s
u
u
w
Ga
Ga
Ga
Ga
Ga
Ga
Ga
Gh
Gh
Gj
Gk
Gl
Gl
Gl
Go
Gp
14
21
Ga
Ga
Gc
Gc
Gc
Gc
Gc
Gh
Gi
Gj
Gk
Gl
Gl
Gn
Go
Gp
16
26
Ga
Gb
Gc
Gd
Gd
Gf
Gg
Gh
Gi
Gj
Gk
Gl
Gm
Gn
Go
Gp
18
33
Ga
Gb
Gc
Gd
Ge
Gf
Gg
Gh
Gi
Gj
Gk
Gl
Gm
Gn
Go
Gp
20
36
s
s
s
s
s
s
s
s
s
Gh
Gi
Gj
Gk
Gl
Gl
Gl
Go
Gp
15
22
s
s
Ga
Ga
Ga
Ga
Ga
Ga
Ga
Gh
Gi
Gj
Gk
Gl
Gl
Gl
Go
Gp
16
24
t
t
Ga
Ga
Ga
Ga
Ga
Ga
Ga
Gh
Gi
Gj
Gk
Gl
Gl
Gn
Go
Gp
17
26
t
t
Ga
Gb
Gc
Gc
Gc
Gc
Gg
Gh
Gi
Gj
Gk
Gl
Gl
Gn
Go
Gp
17
29
v
v
Ga
Gb
Gc
Gc
Gd
Gd
Gg
Gh
Gi
Gj
Gk
Gl
Gl
Gn
Go
Gp
20
33
s
s
Ga
Ga
Ga
Ga
Ga
Ga
Ga
Gh
Gh
Gj
Gk
Gl
Gl
Gl
Go
Gp
17
24
u
u
Ga
Gb
Gc
Gc
Gc
Gc
Gg
Gh
Gi
Gj
Gk
Gl
Gl
Gn
Go
Gp
19
31
617
Table 2. Faunal similarity among sites
618
Jaccard indices of similarity (J) between Solenopsis assemblages from five collection sites at three
619
elevations (1050, 1420 and 2070 m above sea level). Indices were calculated for the 631-bp dataset,
620
and included only those specimens identified on the basis of both morphology (morphospecies) and
621
DNA barcodes
Site 1
1050 m-B
1050 m-B
1050 m-B
1050 m-B
1050 m-C
1050 m-C
1050 m-C
1420 m
1420 m
2070 m-E1
Site 2
1050 m-C
1420 m
2070 m-R1
2070 m-R2
1420 m
2070 m-R1
2070 m-R2
2070 m-R1
2070 m-R2
2070 m-R2
Morphospecies
0.40
0.30
0.14
0.20
0.09
0.17
0.00
0.08
0.10
0.20
10%
0.29
0.36
0.00
0.14
0.09
0.00
0.25
0.00
0.10
0.33
5%
0.38
0.31
0.00
0.13
0.08
0.00
0.20
0.00
0.09
0.33
2%
0.38
0.31
0.00
0.13
0.08
0.00
0.20
0.00
0.09
0.20
1.20%
0.38
0.20
0.00
0.00
0.00
0.00
0.00
0.00
0.08
0.20
622
Fig. 1.
Neighbour-joining tree based on COI sequences (631 bp) of Solenopsis specimens obtained here (S.
623
spp. 1–22) and from GenBank and BOLD (Accession no. and Process ID). Cluster IDs (a–w and Ga–Gp),
624
altitude (1050, 1420 and 2070 m), geographic origin (C, Copalinga; B, Bombuscaro; R1, Reserva Biológica San
625
Francisco-Transect T1; R2, Reserva Biológica San Francisco-NUMEX) and no. of sequences with the same
626
haplotype when more than one (between parentheses) are given. The small tree in the right corner shows
627
neighbour-joining tree based on wg sequences (343 bp). Same symbols are used for corresponding specimens.
628
Fig. 2.
629
310-bp and (C) 631-bp datasets of the COI gene. Arrows indicate local minima identified for each dataset.
630
Fig. 3.
631
Ga–Gp), altitude (1050, 1420 and 2070 m), geographic origin (C, Copalinga; R1, Reserva Biológica San
632
Francisco-Transect T1) and Process ID in BOLD are given.
Proportion of pairwise genetic distances among Solenopsis haplotypes based on the (A) 237-bp, (B)
Bayesian analysis of the concatenated COI and wg datasets. Morphospecies name, cluster IDs (a–w and
Page 19 of 26
C
C
R2
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
Detection of cryptic species by DNA barcoding. (A, B) Some workers of Solenopsis sp. 15 presented
633
Fig. 4.
634
high DNA divergence of the COI gene (>10%) but were not accurately distinguishable. (C, D) Gynes, each one
635
placed below its associated worker, provided new criteria for species identification: longitudinal striae were
636
restricted to the latero-basal part of propodeum in E, whereas they extended above the propodeal spiracle in F
637
(arrows ‘a’), and an anteroventral petiolar tooth was present in E whereas absent in F (arrows ‘b’).
638
Fig. 5.
639
15 based on morphology (‘Complex II’ in Table 1). Each circle represents one haplotype and its size is related
640
to the number of collected individuals. Length of links between circles is proportional to the number of
641
substitutions. Morphospecies (Solenopsis spp. 01, 14 and 15) and genetic clusters (s–w) are indicated.
642
Fig. 6.
643
of the 631-bp dataset. Here, species richness was defined as the number of Solenopsis morphospecies or as the
644
number of clusters obtained using a 10%, 5%, 2% and 1.2% threshold.
645
Haplotype network of 28 COI sequences of 658 bp, from workers identified as Solenopsis spp. 01, 14 or
Estimates of Solenopsis species richness at five sites spread at three altitudes and calculated on the basis
Supplementary material S1. List of 187 Solenopsis specimens selected for DNA analyses
Sample_ID*
Process ID
(BOLD)
Genus
Morphospecies
Caste
33807Ssp141000QD SOLEN024-12 Solenopsis
sp01TD
Worker
33788Ssp5
SOLEN028-12 Solenopsis
sp01TD
Worker
33791Ssp201000QD SOLEN029-12 Solenopsis
sp01TD
Worker
33797Ssp121000QD SOLEN030-12 Solenopsis
sp01TD
Worker
33797Ssp12B
SOLEN031-12 Solenopsis
sp01TD
Worker
33798Ssp121000QD SOLEN032-12 Solenopsis
sp01TD
Worker
33791Ssp6
SOLEN033-12 Solenopsis
sp01TD
Worker
33791Ssp6B
SOLEN035-12 Solenopsis
sp01TD
Worker
33786Ssp171000QD
Solenopsis
sp01TD
Worker
33795Ssp12
Solenopsis
sp01TD
Worker
33795Ssp12B
Solenopsis
sp01TD
Worker
33797Ssp12
Solenopsis
sp01TD
Worker
33799Ssp121000QD
Solenopsis
sp01TD
Worker
33799Ssp171000QD
Solenopsis
sp01TD
Worker
33801Ssp151000QD
Solenopsis
sp01TD
Worker
33801Ssp151000QE
Solenopsis
sp01TD
Worker
33801Ssp151000QF
Solenopsis
sp01TD
Worker
33802Ssp5
Solenopsis
sp01TD
Worker
33802Ssp51000QC
Solenopsis
sp01TD
Worker
Page 20 of 26
Sampling
method
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Sampling s
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
33802Ssp5B
Solenopsis
sp01TD
Worker
33804Ssp12
Solenopsis
sp01TD
Worker
33804Ssp121000QD
Solenopsis
sp01TD
Worker
33804Ssp12B
Solenopsis
sp01TD
Worker
33809Ssp12
Solenopsis
sp01TD
Worker
33809Ssp121000QD
Solenopsis
sp01TD
Worker
33809Ssp201000QD
Solenopsis
sp01TD
Worker
33809Ssp5
Solenopsis
sp01TD
Worker
33809Ssp51000Q
Solenopsis
sp01TD
Worker
33809Ssp51000QB
Solenopsis
sp01TD
Worker
33809Ssp51000QC
Solenopsis
sp01TD
Worker
33809Ssp51000QD
Solenopsis
sp01TD
Worker
33809Ssp5B
Solenopsis
sp01TD
Worker
33810Ssp171000QD
Solenopsis
sp01TD
Worker
33791Ssp61000Q
SOLEN034-12 Solenopsis
sp02TD
Worker
33801Ssp6
SOLEN036-12 Solenopsis
sp02TD
Worker
33803Ssp61000Q
SOLEN037-12 Solenopsis
sp02TD
Worker
33788Ssp61000Q
Solenopsis
sp02TD
Worker
33802Ssp6
Solenopsis
sp02TD
Worker
33802Ssp61000Q
Solenopsis
sp02TD
Worker
33802Ssp6B
Solenopsis
sp02TD
Worker
33791Ssp14
SOLEN038-12 Solenopsis
sp07TD
Worker
33802Ssp14
SOLEN039-12 Solenopsis
sp07TD
Worker
33807Ssp141000QE
Solenopsis
sp07TD
Worker
33808Ssp121000QD
Solenopsis
sp07TD
Worker
33809Ssp14
Solenopsis
sp07TD
Worker
33809Ssp14B
Solenopsis
sp07TD
Worker
33768Ssp41000QC SOLEN026-12 Solenopsis
sp15TD
Worker
33768Ssp4
sp15TD
Worker
SOLEN040-12 Solenopsis
Page 21 of 26
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
33786Ssp4
SOLEN041-12 Solenopsis
sp15TD
Worker
33795Ssp121000QD SOLEN042-12 Solenopsis
sp15TD
Worker
33803ASsp16
SOLEN043-12 Solenopsis
sp15TD
Worker
33803BSsp16
SOLEN044-12 Solenopsis
sp15TD
Worker
33803CSsp16
SOLEN045-12 Solenopsis
sp15TD
Worker
33805Ssp4
SOLEN046-12 Solenopsis
sp15TD
Worker
33796Ssp181000QD
Solenopsis
sp16TD
Worker
33801Ssp181000QD
Solenopsis
sp16TD
Worker
33810Ssp181000QD
Solenopsis
sp16TD
Worker
3380101sp16TD
Solenopsis
sp16TD
Worker
3381001sp16TD
Solenopsis
sp16TD
Worker
4028902sp01TD
Solenopsis
sp01TD
Gyne
3463505sp01TD
Solenopsis
sp01TD
Worker
3463506sp01TD
Solenopsis
sp01TD
Worker
3463508sp01TD
Solenopsis
sp01TD
Worker
3515615sp01TD
Solenopsis
sp01TD
Worker
3515615sp01TD
Solenopsis
sp01TD
Worker
3516422sp01TD
Solenopsis
sp01TD
Worker
3516422sp01TD
Solenopsis
sp01TD
Worker
35336182sp01TD
Solenopsis
sp01TD
Worker
3533618sp01TD
Solenopsis
sp01TD
Worker
3533808sp01TD
Solenopsis
sp01TD
Worker
3533808sp01TD
Solenopsis
sp01TD
Worker
4022327sp01TD
Solenopsis
sp01TD
Worker
4022327sp01TD
Solenopsis
sp01TD
Worker
4026827sp01TD
Solenopsis
sp01TD
Worker
4026827sp01TD
Solenopsis
sp01TD
Worker
4028417sp01TD
Solenopsis
sp01TD
Worker
4028417sp01TD
Solenopsis
sp01TD
Worker
4129818sp01TD
SOLEN001-12 Solenopsis
sp01TD
Gyne
Page 22 of 26
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
Winkler
(1050m-B
Bombuscaro - Podocarp
Winkler
(1050m-B
Bombuscaro - Podocarp
Winkler
(1050m-B
Bombuscaro - Podocarp
Winkler
(1050m-B
Bombuscaro - Podocarp
Winkler
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Copalinga Private Reserve
Queen
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
C)
Nest found in Copalinga Private Reserve
dead wood
C)
Nest found in Copalinga Private Reserve
dead wood
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
C)
Winkler
Copalinga Private Reserve
Winkler
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
3463504sp01TD
SOLEN002-12 Solenopsis
sp01TD
Worker
4127214sp01TD
SOLEN006-12 Solenopsis
sp01TD
Gyne
4029009sp01TD
SOLEN010-12 Solenopsis
sp01TD
Gyne
4128010sp01TD
SOLEN011-12 Solenopsis
sp01TD
Gyne
3463507sp01TD
SOLEN025-12 Solenopsis
sp01TD
Worker
3464908sp01TD
SOLEN047-12 Solenopsis
sp01TD
Worker
4026826sp01TD
SOLEN048-12 Solenopsis
sp01TD
Worker
4027026sp01TD
SOLEN049-12 Solenopsis
sp01TD
Worker
4027028sp01TD
SOLEN050-12 Solenopsis
sp01TD
Worker
3653105sp02TD
Solenopsis
sp02TD
Worker
3653104sp02TD
SOLEN023-12 Solenopsis
sp02TD
Worker
3653106sp02TD
SOLEN051-12 Solenopsis
sp02TD
Worker
3534109sp06TD
SOLEN052-12 Solenopsis
sp06TD
Worker
4030215sp08TD
Solenopsis
sp08TD
Worker
4030215sp08TD
Solenopsis
sp08TD
Worker
4660902sp22TD
SOLEN012-12 Solenopsis
sp22TD
Worker
4038719sp01TD
SOLEN013-12 Solenopsis
sp01TD
Worker
4111406_2sp01TD SOLEN053-12 Solenopsis
sp01TD
Worker
4111406sp01TD
SOLEN054-12 Solenopsis
sp01TD
Worker
4038719sp01TD
4039526sp01TD
4038215sp07TD
4035116sp14TD
4045308sp14TD
4034412sp14TD
4034811sp14TD
4040310sp14TD
4038623sp14TD
4035120sp14TD
4038226sp14TD
4039527sp14TD
4039528sp14TD
4034411sp14TD
4034413sp14TD
4035012sp14TD
4037219sp14TD
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
Solenopsis
sp01TD
sp01TD
sp07TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
sp14TD
Worker
Worker
Worker
Gyne
Worker
Worker
Worker
Worker
Worker
Worker
Worker
Worker
Worker
Worker
Worker
Worker
Worker
SOLEN055-12
SOLEN004-12
SOLEN005-12
SOLEN014-12
SOLEN015-12
SOLEN016-12
SOLEN027-12
SOLEN056-12
SOLEN057-12
SOLEN058-12
SOLEN059-12
Page 23 of 26
C)
Nest found in Copalinga Private Reserve
dead wood
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
Winkler
C)
Nest found in Copalinga Private Reserve
dead wood
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
C)
Nest found in Copalinga Private Reserve
dead wood
C)
Nest found in Copalinga Private Reserve
dead wood
C)
Nest found in Copalinga Private Reserve
dead wood
C)
Copalinga Private Reserve
Winkler
C)
Copalinga Private Reserve
C)
Visual search,
arboreal
species
Copalinga Private Re
Winkler
Copalinga Private Reserve
Nest found in
dead wood Copalinga Private Reserve
Nest found in
dead wood Copalinga Private Reserve
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Winkler
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
4038818sp14TD
Solenopsis
sp14TD
Worker
4045210sp14TD
Solenopsis
sp14TD
Worker
4041203sp15TD
SOLEN007-12 Solenopsis
sp15TD
Gyne
Winkler
Nest found in
4111002sp15TD
SOLEN008-12 Solenopsis
sp15TD
Gyne
dead wood
4037905sp15TD
SOLEN017-12 Solenopsis
sp15TD
Worker
Winkler
Nest found in
4111005_3sp15TD SOLEN060-12 Solenopsis
sp15TD
Worker
dead wood
4036322sp15TD
SOLEN061-12 Solenopsis
sp15TD
Worker
Winkler
4040015sp15TD
SOLEN062-12 Solenopsis
sp15TD
Worker
Winkler
4044510sp15TD
SOLEN063-12 Solenopsis
sp15TD
Worker
Winkler
Nest found in
4110107sp15TD
SOLEN064-12 Solenopsis
sp15TD
Worker
dead wood
Nest found in
4110108sp15TD
SOLEN065-12 Solenopsis
sp15TD
Worker
dead wood
Nest found in
4111005_2sp15TD SOLEN066-12 Solenopsis
sp15TD
Worker
dead wood
Nest found in
4111005sp15TD
SOLEN067-12 Solenopsis
sp15TD
Worker
dead wood
4038212sp16TD
SOLEN009-12 Solenopsis
sp16TD
Male
Winkler
4039608sp16TD
SOLEN018-12 Solenopsis
sp16TD
Gyne
Winkler
4039522sp16TD
SOLEN068-12 Solenopsis
sp16TD
Worker
Winkler
4039523sp16TD
SOLEN069-12 Solenopsis
sp16TD
Worker
Winkler
4039524sp16TD
SOLEN070-12 Solenopsis
sp16TD
Worker
Winkler
4042106sp17TD
Solenopsis
sp17TD
Worker
4037710_2sp18TD SOLEN071-12 Solenopsis
sp18TD
Worker
Winkler
4037710sp18TD
SOLEN072-12 Solenopsis
sp18TD
Worker
Winkler
4039112sp19TD
SOLEN003-12 Solenopsis
sp19TD
Worker
Winkler
4037517sp20TD
Solenopsis
sp20TD
Worker
4036323sp21TD
SOLEN019-12 Solenopsis
sp21TD
Worker
Winkler
4049004sp01TD
SOLEN020-12 Solenopsis
sp01TD
Gyne
4048807sp01TD
SOLEN073-12 Solenopsis
sp01TD
Worker
4053106sp01TD
SOLEN074-12 Solenopsis
sp01TD
Worker
4054604sp01TD
SOLEN075-12 Solenopsis
sp01TD
Worker
4131005sp01TD
SOLEN077-12 Solenopsis
sp01TD
Worker
4134704sp01TD
SOLEN078-12 Solenopsis
sp01TD
Worker
33662Ssp102000QC
Solenopsis
sp11TD
Worker
33688Ssp10
Solenopsis
sp11TD
Worker
33688Ssp10B
Solenopsis
sp11TD
Worker
33690Ssp10
Solenopsis
sp11TD
Worker
33709Ssp10
Solenopsis
sp11TD
Worker
33709Ssp10B
Solenopsis
sp11TD
Worker
33710Ssp10
Solenopsis
sp11TD
Worker
Page 24 of 26
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Copalinga Private Reserve
Reserva Biológica San Fr
Winkler
(2070m-R
Reserva Biológica San Fr
Winkler
(2070m-R
Reserva Biológica San Fr
Winkler
(2070m-R
Reserva Biológica San Fr
Winkler
(2070m-R
Nest found in Reserva Biológica San Fr
dead wood
(2070m-R
Nest found in Reserva Biológica San Fr
dead wood
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
4266506sp11TD
SOLEN021-12 Solenopsis
sp11TD
Gyne
4051712sp11TD
SOLEN079-12 Solenopsis
sp11TD
Worker
4051713sp11TD
SOLEN080-12 Solenopsis
sp11TD
Worker
4053010sp11TD
SOLEN081-12 Solenopsis
sp11TD
Worker
4053011sp11TD
SOLEN082-12 Solenopsis
sp11TD
Worker
4053012sp11TD
SOLEN083-12 Solenopsis
sp11TD
Worker
4052604_2sp12TD SOLEN084-12 Solenopsis
sp12TD
Worker
4052604sp12TD
SOLEN085-12 Solenopsis
sp12TD
Worker
4053101sp13TD
SOLEN022-12 Solenopsis
sp13TD
Worker
4130805sp01TD
SOLEN076-12 Solenopsis
sp13TD
Worker
33692Ssp72000Q
Solenopsis
sp15TD
Worker
33700Ssp72000Q
Solenopsis
sp15TD
Worker
33701Ssp72000Q
Solenopsis
sp15TD
Worker
33710_1
Solenopsis
sp15TD
Worker
33710_2
Solenopsis
sp15TD
Worker
33688Ssp102000QC SOLEN086-12 Solenopsis
sp11TD
Worker
33688Ssp102000QD SOLEN087-12 Solenopsis
sp11TD
Worker
33690Ssp102000QC SOLEN088-12 Solenopsis
sp11TD
Worker
33690Ssp102000QD SOLEN089-12 Solenopsis
sp11TD
Worker
33709Ssp102000QC SOLEN090-12 Solenopsis
sp11TD
Worker
33709Ssp102000QD SOLEN091-12 Solenopsis
sp11TD
Worker
33710Ssp102000QC SOLEN092-12 Solenopsis
sp11TD
Worker
33688Ssp7
SOLEN093-12 Solenopsis
sp15TD
Worker
33688Ssp72000Q
SOLEN094-12 Solenopsis
sp15TD
Worker
33696Ssp72000Q
SOLEN095-12 Solenopsis
sp15TD
Worker
33702Ssp9
SOLEN096-12 Solenopsis
sp15TD
Worker
33703Ssp7
SOLEN097-12 Solenopsis
sp15TD
Worker
33703Ssp72000Q
SOLEN098-12 Solenopsis
sp15TD
Worker
33705Ssp7
SOLEN099-12 Solenopsis
sp15TD
Worker
33707Ssp7
SOLEN100-12 Solenopsis
sp15TD
Worker
Page 25 of 26
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Soil sample
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Winkler
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
(2070m-R
Reserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
Publisher: CSIRO; Journal: IS:Invertebrate Systematics
Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030
DOI: 10.1071/IS12030; TOC Head:
646
Winkler
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San Fr
T1 (2070mReserva Biológica San F
T1 (2070mReserva Biológica San Fr
T1 (2070m-
Worker
Pitfall 24h
Paraguay: Boqueron Fo
sp. 01(EC)TD
Worker
Winkler
SOLEN108-12 Strumigenys
sp. 03(EC)TD
Worker
Winkler
SOLEN107-12 Wasmannia
auropunctata
Worker
Winkler
33707Ssp72000Q
SOLEN101-12 Solenopsis
sp15TD
Worker
33708Ssp7
SOLEN102-12 Solenopsis
sp15TD
Worker
33708Ssp72000Q
SOLEN103-12 Solenopsis
sp15TD
Worker
33709Ssp9
SOLEN104-12 Solenopsis
sp15TD
Worker
33710Ssp7
SOLEN105-12 Solenopsis
sp15TD
Worker
33710Ssp9
SOLEN106-12 Solenopsis
sp15TD
Worker
434202spgem01
SOLEN110-12 Solenopsis
sp.01 (PAG) nr
geminata
33806Ssp11000Q
SOLEN109-12 Strumigenys
33803Psp31000
33788Wa1000
*
In BOLD, specimens deposited have their Sample ID preceded by SOLEN.
Page 26 of 26
Winkler
Winkler
Winkler
Winkler
Winkler
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B
Bombuscaro - Podocarp
(1050m-B