High species turnover of Solenopsis (Hymenoptera ... · Web viewHigh species turnover of the ant genus Solenopsis (Hymenoptera : Formicidae) along an altitudinal gradient in the

High species turnover of Solenopsis (Hymenoptera: Formicidae) along an altitudinal gradient in the Ecuadorian Andes revealed (

Publisher: CSIRO; Journal: IS:Invertebrate Systematics

Article Type: research-article; Volume: ; Issue: ; Article ID: IS12030

DOI: 10.1071/IS12030; TOC Head:



DOI: 10.1071/IS12030; TOC Head:



DOI: 10.1071/IS12030; TOC Head:

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

Thibaut DelsinneA,C, Gontran SonetB, Zoltán T. NagyB, Nina WautersA, Justine JacqueminA and Maurice LeponceA

ABiological Evaluation Section, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000 Brussels, Belgium.

BJoint Experimental Molecular Unit, Royal Belgian Institute of Natural Sciences, Rue Vautier 29, B-1000 Brussels, Belgium.

CCorresponding author. Email: [email protected]

Solenopsis is a widespread ant genus and the identification of its species is notoriously difficult. Hence, investigation of their distribution along elevational gradients is challenging. Our aims were (1) to test the complementarity of the morphological and DNA barcoding approaches for Solenopsis species identification, and (2) to assess species diversity and distribution along an altitudinal gradient in the Ecuadorian Andes. Ants were collected in five localities between 1000 and 3000 m above sea level. In total, 24 morphospecies were identified along the gradient and 14 of them were barcoded. Seven morphospecies were confirmed by the molecular approach. Three others, occurring sympatrically and possessing clear diagnostic characters, showed low genetic divergence. Representatives of a further four morphospecies were split into nine clusters by COI and nuclear wingless genetic markers, suggesting the existence of cryptic species. Examination of gynes revealed potential diagnostic characters for morphological discrimination. Solenopsis species were found up to an altitudinal record of 3000 m. Most morphospecies (20 of 24) were found at a single elevation. Our results suggest a high species turnover along the gradient, and point to the use of morphological and DNA barcoding approaches as necessary for differentiating among Solenopsis species.

IS12030

High altitudinal species turnover of Solenopsis ants.

T. Delsinne et al.

Manuscript received 18 April 2012, accepted 16 September 2012, published online dd mmm yyyy

Introduction

Solenopsis Westwood, 1840 is a large myrmicine ant genus encompassing 183 species, with a worldwide distribution (Guénard et al. 2010; Bolton 2012). The most well known species of this genus inflict a painful sting and are known as fire ants. Some fire ants, such as Solenopsis invicta, have become important invasive pests (Tschinkel 2006). Other species are referred to as thief ants because some of them are known to steal food from other ants (Pacheco 2007). About half of all described Solenopsis species are found in the Neotropical region (Fernández and Sendoya 2004). Solenopsis nests can be found virtually everywhere, in soil, leaf litter, dead wood, epiphytes or plant cavities (Creighton 1950). Workers forage from deep in the soil (Ryder Wilkie et al. 2007) to high in the forest canopy (Blüthgen et al. 2000). They are one of the most frequently encountered ant genera in ground-dwelling ant communities (Ward 2000; Donoso and Ramón 2009; Braga et al. 2010) and, as a consequence of their diversity and abundance, they are considered to be of significant ecological importance in the Neotropics (Ward 2000).

Members of the genus can be easily differentiated from other Myrmicinae by the 10-segmented antenna with a 2-jointed club, the propodeum rounded and unarmed, the petiole and postpetiole nodes well developed, and the clypeus longitudinally bicarinate with an isolated median seta (Ettershank 1966; Bolton 2003). Identification to specific level is, however, extremely difficult. Mackay and Mackay (2002) indicated that ‘identification is nearly impossible’. Creighton (1930), in his incomplete revision of the New World Solenopsis, wrote: ‘Carlo Emery once characterised the genus Solenopsis as the crux myrmecologorum. That the term is apt no one who has experienced the difficulties of the group will deny, least of all the author who, at the end of three years of study, still finds the «cross» a heavy burden’. In fact, the literature abounds with quotations describing similar opinions. For instance: ‘the genus Solenopsis is no favorite of ant taxonomists’ (Thompson 1989), ‘at least some of [Solenopsis] are exceedingly difficult to classify’ (Smith 1943), ‘the members of the thief ant group of the genus Solenopsis have had a notorious reputation of being difficult to identify for over 70 years … this reputation is merited’ (Pacheco 2007). Snelling (2001) indicated, while describing S. maboya, a new thief ant from Puerto Rico, that ‘maboya is the Taino (Arawak) word for a perverse spirit, and seemed appropriate, given the challenging nature of the taxonomy of this group of ants’.

The difficulty of identifying specimens of Solenopsis is explained by two factors. First, worker morphology monotonously lacks diagnostic characters. Thief ants are tiny, often less than 2 mm long, which complicates the recognition of morphological characters. Fire ants are larger but polymorphic, presenting a continuum of sizes in the same nest. For instance, workers of S. invicta range from 2.65 mm to 6.16 mm in body size (Tschinkel et al. 2003). Moreover, all species of fire ants and several thief ant species exhibit intraspecific variation in morphological traits which may exceed interspecific differences (Pitts et al. 2005; Pacheco 2007; Ross et al. 2010). Males and gynes may be less uniform morphologically and offer additional characters for species identification (Creighton 1950). However, these reproductive castes are less frequently encountered and rarely associated with workers (Creighton 1950; Pacheco 2007). Second, most species were inadequately described on the basis of limited material, mainly between the end of the 19th and beginning of the 20th centuries. The use of numerous trinomials and quadrinomials has generated serious taxonomical confusion (Pacheco 2007). Creighton (1930) attempted to revise New World Solenopsis but most thief ants were not included in his work since he planned to treat them in a separate publication which never eventuated. Trager (1991) restricted his revision to fire ants but, even afterwards, species delimitation often remains problematic (Ross et al. 2010). More recently, Pacheco (2007) revised New World thief ants, proposed eight species complexes and numerous new synonymies and other changes, recognised 83 species and presented keys for the identification of workers. Unfortunately, this thesis does not conform to the rules of the International Code of Zoological Nomenclature, making it impossible to acknowledge his taxonomical changes (William Mackay, pers. comm.).

Thus, the α-taxonomy of Solenopsis is still confused, which represents a serious impediment for ant biodiversity inventories, and ecological work in general. Most collected species are misidentified (Thompson 1989) or are simply recorded only as morphotypes. For instance, 13 Solenopsis species were sampled in the Nouragues Research Station, in French Guiana, but only two of them could be assigned to a valid name (Groc et al. 2009). Similarly, only one of 15 species collected during a thorough inventory in Ecuadorian Amazonian forests was named (Ryder Wilkie et al. 2010).

Recently, the use of DNA barcodes, short mitochondrial DNA sequences of the cytochrome oxidase I (COI) gene, has been proposed to facilitate species identification and discovery (Hebert et al. 2003; Janzen et al. 2009). This method is acknowledged as a useful explorative tool to provide estimates of species numbers, especially in very diverse and poorly understood taxonomic groups (Wiemers and Fiedler 2007; Jansen et al. 2009; Strutzenberger et al. 2011; Tänzler et al. 2012). In particular, DNA barcoding has proved useful in complementing morphological species determination in biodiversity surveys of ants (Smith et al. 2005; Fisher et al. 2008; Fisher and Smith 2009), in facilitating caste association (Fisher et al. 2008), and assisting in the discovery of new cryptic ant species (Schlick-Steiner et al. 2006; Fisher et al. 2008; Menke et al. 2010). Nevertheless, the barcoding approach possesses several pitfalls and shortcomings (reviewed in Rubinoff et al. 2006; Jinbo et al. 2011) with barcoding success rate varying among taxa (Elias et al. 2007; Jansen et al. 2009; Wild 2009). Therefore, species hypotheses based on DNA barcodes should be supported by additional, independent nuclear markers (Ross et al. 2010; Smith et al. 2011) or other data such as morphology, geography, ecology or behaviour (Yassin et al. 2010).

For unknown reasons, a previous attempt to amplify the COI marker from thief ants was not successful (Pacheco 2007) and, so far, most genetic studies of Solenopsis have focussed on fire ants (Ross and Shoemaker 2005; Shoemaker et al. 2006; Ross et al. 2010). These analyses have identified genetically independent lineages within variable and widespread taxa. Further, the combined use of mitochondrial and nuclear markers have revealed cryptic species (Ross et al. 2010).

Identification of Solenopsis species is expected to be particularly challenging along elevational gradients, where it is frequently found that ants once considered to belong to a single widely distributed species turned out to be several cryptic species with parapatric distributions and restricted altitudinal ranges (Lattke 2003). Our aims in this study were (1) to test the complementarity of morphological and DNA barcoding approaches for species identification in the genus Solenopsis, and (2) to assess species diversity and distribution along an altitudinal gradient in the Ecuadorian Andes, which is considered to be a biodiversity hotspot for many taxa.

Materials and methods

Ant sampling

Ants were collected between 2007 and 2011 in seven forested sites spread among five localities between 1000 m and 3000 m above sea level with elevational steps of 500 m between localities. Study sites were selected in the Podocarpus National Park and two adjacent protected areas (Reserva Biológica San Francisco and Copalinga private reserve), on the eastern range of the South Ecuadorian Andes, in the provinces of Loja and Zamora-Chinchipe. Details about the study area are provided in Beck et al. (2008). Five reference sites were selected: Copalinga Private Reserve –blue trail (called hereafter ‘1050 m-C’; 4°5S, 78°57(W), Copalinga Private Reserve – red trail (‘1420 m’; 4°5S, 78°58(W); Reserva Biológica San Francisco – Transect T1 (‘2070 m-R1’; 3°58(S, 79°5W), El Tiro (‘2500 m’; 3°59(S, 79°7(W) and Cajanuma-Podocarpus National Park (‘3000 m’; 4°6(S, 79°10(W). Two supplementary sites were sampled at 1050 and 2070 m: Bombuscaro-Podocarpus National Park (‘1050 m-B’; 4°6(S, 78°58(W) and Reserva Biológica San Francisco-NUMEX (‘2070 m-R2’; 3°58(S, 79°4(W). The distance between sites ranged from 2 to 20 km. At each site, ants present in quadrats of leaf litter were extracted by the Winkler method (54 m2 extracted per site). In addition, we searched for Solenopsis nests in dead wood, soil and vegetation in an attempt to document reproductive castes in association with series of workers. Specimens were preserved in 96% ethanol (denatured with diethyl ether) and sorted to morphospecies on the basis of criteria proposed by Pacheco (2007), such as expression of clypeal teeth, number of ommatidia, number of mandibular teeth, scape length, body colour, pattern and extent of sculpture, shape and size of body tagma, expression of anteroventral petiolar process and size of cephalic punctures. We used the phenetic species concept and expected that a certain degree of difference in morphological characters indicated potential reproductive isolation.

A few specimens from each morphospecies were pinned and photographed. Images were taken with a Leica DFC290 camera attached to a Leica Z6APO stereomicroscope. A series of images was taken by focusing on different levels of the insect, using the Leica Application Suite v38 (2003–2011) and combined with CombineZP (Hadley 2010). Final processing of images was done in Adobe Photoshop CS5. Original images were deposited in Morphbank (collection no.: 801203; http://www.morphbank.net/801203). Voucher specimens were deposited at the Royal Belgian Institute of Natural Sciences, Brussels, Belgium, and at the Universidad Técnica Particular de Loja, Loja, Ecuador.

Laboratory method

About 10 260 Solenopsis specimens were collected during this study (up to 4605 specimens per site). Multiple representatives (n = 2–70) of each of the most abundant Solenopsis morphospecies were selected for DNA analysis. Seven morphospecies represented by fewer than five individuals were discarded. Analyses were carried out on 187 Solenopsis specimens (Supplementary material S1). Total genomic DNA was isolated from the complete ant body using the commercial NucleoSpin Tissue Kit (Macherey-Nagel, Germany). After DNA extraction, specimens were preserved as vouchers in absolute ethanol. Amplification of the mitochondrial cytochrome c oxidase subunit I (COI) marker was carried out in polymerase chain reaction (PCR) using the primer pair LF1 and LR1 (Smith et al. 2005) modified from Hebert et al. (2004a) and the universal primers LCO1490 and HCO2198 (Folmer et al. 1994). When amplification systematically failed, DNA quality was checked on 1.2% agarose gel, and smaller DNA fragments were amplified using the primer combination LCO1490 and LCO-ANTMR1D-RonIIdeg_R (Fisher and Smith 2008) modified from Simon et al. (1994). Amplification of the nuclear wingless (wg) marker was performed for a selection of 1–3 sample(s) per COI haplogroup using primers wg578F (Ward and Downie 2005) and wg1032R (Abouheif and Wray 2002). Each PCR was prepared in a total volume of 25 µL containing 2 µL of DNA template and 0.03 U µL–1 of Platinum® Taq DNA polymerase (Life Technologies, USA), 1( PCR buffer, 0.2 mm dNTPs, 0.4 μm of each primer, 1.5 mm MgCl2. PCR protocol followed the profile of 94°C for 3 min; 5 cycles 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 for 60 s; followed by a terminal elongation step at 72°C for 7 min, and subsequent storage at 4°C. PCR products were visualised on ~1.2% agarose gel, and purified using the NucleoFast 96 PCR Plate (Macherey-Nagel, Germany). PCR products were sequenced with an ABI 3130xl automated capillary sequencer using BigDye v1.1 chemistry and following the manufacturer’s instructions (Life Technologies, USA).

Genetic data analysis

DNA sequences were checked for quality and aligned by hand. No internal stop codons were detected. Homologous fragments of COI sequences of Solenopsis available in GenBank and BOLD (Ratnasingham and Hebert 2007) were added to the dataset provided that no characters were missing. As the length of the sequences obtained varied from 237 to 658 bp, three datasets were created: one including a maximum number of samples but with short sequences (237 bp) and two with longer sequences but including fewer samples (310 and 631 bp). Distributions of pairwise uncorrected distances were plotted for all genetic datasets using the R language and environment for statistical computing and graphics ver. 2.14.2 (R developmental core team) and package ape v2.7-3 (Paradis et al. 2004). For an overview of the pairwise genetic distances, a neighbour-joining tree with bootstrapping (1000 replicates) was constructed on the basis of the uncorrected distance matrix of the 631-bp dataset and using MEGA v5.01 (Tamura et al. 2011). Putative species delimitation was performed using uncorrected distances without a phylogenetic tree reconstruction in which the assumption of monophyly would be doubtful based on a single gene and incomplete sampling (Taylor and Harris 2012). In the absence of species identifications, intraspecific distances could not be distinguished from interspecific distances and no optimal threshold distance could be defined for species delimitation. For this reason, different threshold values were used. Since intraspecific distances are expected to be generally lower than interspecific distances – forming a ‘barcoding gap’ – (Hebert et al. 2004b), local minima in the distribution of genetic distances can be used as tentative threshold distance to test for delineation of species. All local minima of the density of the pairwise distances were determined using the function localMinima of package spider v1.1-2 (Brown et al. 2012) and were used as thresholds. On the basis of the literature, we also selected threshold values of 2% and 10%. The former was proposed as a standard distance for ants (Smith et al. 2005; Smith and Fisher 2009) and the latter represented an extreme value rarely surpassed by intraspecific distances (e.g. Smith and Fisher 2009; Yassin et al. 2010).

Clustering of samples was performed for each threshold and based on pairwise distances using the function tclust of the package spider v1.1-2 (Brown et al. 2012). Samples showing genetic distances greater than the threshold with any member of a cluster were excluded from that cluster. For the cluster encompassing more than five different haplotypes, a haplotype network was calculated with pegas v0.4-1 (Paradis 2010) based on the longest fragment available for this subset of samples (658 bp). Finally, a tree was calculated with the Bayesian method of phylogenetic inference, and based on the available concatenated sequences of COI (658 bp) and wg (343 bp).

The dataset was partitioned into six, each partition representing separated codon positions. The Bayesian information criterion implemented in jModeltest v0.1.1 (Posada 2008) was used to find appropriate nucleotide substitution models for all partitions, and recommended settings were used in the subsequent Bayesian analysis. Analysis was carried out with MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) running 10 million generations in two runs. Each run involved four Monte Carlo Markov chains, one of them being cold and the three others heated using MrBayes’ default settings. Every 1000th generation was sampled. Split frequencies were observed, and the convergence of the chains was monitored by Tracer v1.5 (Rambaut and Drummond 2009). At the end of the run, the potential scale reduction factor was checked for all parameters, and was found to be close to 1.0. The first 25% of the sampled trees were discarded (‘burn in’) and the remaining trees were used to construct a consensus. COI and wg sequences were deposited in BOLD (Ratnasingham and Hebert 2007) with Process ID from SOLEN001–12 to SOLEN110–12.

Faunal similarity among sites

The faunal similarity of Solenopsis species among sites was compared using the Jaccard index of similarity (J): J = A/(A+B+C), where for any Sites 1 and 2, A is the number of species present in both sites, B the number of species only at Site 1 and C the number of species only at Site 2. Hence, when J = 1, both sites host the same Solenopsis species, and when J = 0, Solenopsis species collected in the two sites are completely different. We restricted this analysis to the dataset containing the longest (631 bp) sequences. Only specimens documented by both morphological and DNA barcode data were included. We compared matrices of similarity obtained with the morphological and DNA barcoding approaches by Mantel tests (24 iterations) using Mantel 2.0 (Liedloff 1999).

Results

Species delimitation and DNA barcoding

Overall, 24 morphospecies of Solenopsis were identified in our sampling in the Ecuadorian Andes. Seven of these morphospecies were rare (1–5 individuals) and not used for genetic analyses. Microscopic examination of voucher specimens after DNA extraction confirmed that most anatomical features useful for species determination were preserved (see extracted specimens on Figs S8, S17-S19 in Supplementary material S2). COI sequences were obtained for 106 specimens representing 14 morphospecies (no sequence was obtained for three of the morphospecies selected). Their lengths varied from 198 to 658 bp. Among them, 19 shorter sequences were obtained with the alternative primer combination: LCO-ANTMR1D-RonIIdeg_R. Datasets, including GenBank and BOLD sequences, consisted of (1) 245 sequences of 237 bp, representing 54 haplotypes, (2) 245 sequences of 310 bp, representing 55 haplotypes, and (3) 213 sequences of 631 bp, representing 54 haplotypes.

The DNA barcoding approach allowed us to successfully associate seven gynes and one male to the worker caste (identical sequences) for seven of the clusters separated by minimum 5%. In one cluster (Solenopsis sp. 16), worker, male and gyne castes were documented.

The neighbour-joining tree based on the COI sequences of 631 bp (Fig. 1) showed genetic distance between haplotypes (from 0 to 21.4%). Local minima in the distributions of pairwise distances (p distance) were between 1.2 and 10% depending on the dataset (Fig. 2). Using these different thresholds, 21–36 clusters of similar sequences were defined for the entire dataset and, excluding GenBank and BOLD sequences, 14–20 clusters were defined for Solenopsis collected in Ecuador (Table 1). Sequences of the nuclear wg gene fragment (343 bp) were obtained for 26 Solenopsis workers. They represented 12 haplotypes and 11–12 clusters defined with COI using 5–1.2% distance threshold, respectively. Genetic divergences among these nuclear sequences varied from 0 to 7% (Fig. 1, small tree). Before delimiting species based on COI sequences, we verified that no conflicts were observed between the wg and the COI trees and detected no sign of hybridisation or other horizontal gene transfer. Bayesian analysis of the concatenated COI–wg dataset resolved most of the nodes for 10–11 Solenopsis clusters (defined with COI using 5–1.2% distance treshold: Fig. 3). Three morphospecies (Solenopsis spp. 01, 14 and 15) that were morphologically similar were not sister species.

All sequences obtained for COI from Ecuadorian Solenopsis specimens diverged considerably from sequences available in GenBank and BOLD (p distance >9.2%). Five situations were observed:

(a) Seven clusters were consistently discriminated using any threshold value and perfectly matched morphospecies classification (‘Perfect match’ in Table 1).

(b) Seven other clusters were also consistently discriminated using any threshold value but corresponded to four morphospecies (‘Splitting’ in Table 1). A thorough reexamination of specimens recognised reliable diagnostic criteria for two of these genetically well defined clusters (previously identified as Solenopsis sp. 01 [Fig. S12 in Supplementary material S2] and Solenopsis spp. 14–21 [Fig. S15 in Supplementary material S2]). The last five clusters corresponded to ants presenting variation in their morphology but for which no reliable diagnostic criteria could be identified (Supplementary material S2).

(c) Three morphospecies presented clearly distinctive and consistent morphology and corresponded to three genetically closely related groups (2%) of ants collected in the same locality at 2070 m (‘Complex I’ in Table 1). The first one (Solenopsis sp. 11) included brown ants whose head, mesosoma, petiole and postpetiole were uniformly foveate (Fig. S8 in Supplementary material S2). The second one (Solenopsis sp. 12) corresponded to yellow ants with smooth head and pronotum and foveate mesonotum, propodeum, petiole and postpetiole (Fig. S10 in Supplementary material S2). Ants from the third morphospecies (Solenopsis sp. 13) were entirely smooth and pale yellow (Fig. S9 in Supplementary material S2).

(d) In contrast, a deep genetic divergence (>10%) was found among workers of morphospecies 15 (Table 1, Fig. 4A, B). A close examination of gyne morphology – associated with workers by barcoding – supported the hypothesis that Solenopsis sp. 15 contains at least two cryptic species (Fig. 4C–F).

(e) Lastly, specimens identified as Solenopsis spp. 01, 14 and 15 presented variable morphology (e.g. body size, colour) and were genetically grouped together (‘Complex II’ in Table 1). In this case, clustering based on DNA and morphology were not consistent. The haplotype network of 28 sequences of 658 bp showed that the observed genetic variation was related to elevation (Fig. 5). No haplotypes were shared between specimens found at different altitudes or at the same altitude but different sites. Furthermore, haplotypes found at lower (1050 m) or higher (2070 m) altitudes were always connected with haplotypes found at intermediate elevation (1420 m).

Diversity and distribution of Solenopsis

Both morphological and DNA barcoding approaches suggested a clear disparity among sites in terms of species composition (Table 1). On the basis of morphology, only two morphospecies (Solenopsis spp. 01 and 15) were found at 1050, 1420 and 2070 m, and two (Solenopsis spp. 07 and 16) at 1050 and 1420 m. Solenopsis spp. 01 and 15 belonged to the same complex (‘Complex II’ in Table 1), which possessed genetic variation related to elevation (Fig. 5). The presence of Solenopsis sp. 16 at ‘1050 m-B’ could not be confirmed by DNA barcoding because no DNA sequences were obtained from specimens of the corresponding morphospecies from this elevation. Except for these four taxa, all morphospecies and well discriminated genetic lineage (p distance >5%) were restricted to a single altitude (Table 1). As a result, similarity of Solenopsis assemblages among sites was low. For instance, with the morphological approach, the Jaccard index of similarity (J) ranged from 0 to 0.4, and with the DNA barcoding approach, from 0 to 0.38 (using a 5% threshold and the 631 bp dataset) (Table 2). Patterns of assemblage similarity among sites obtained with morphology and DNA barcoding were similar, and not dependant on the threshold (Table 2) (Mantel tests, 24 iterations, 0.85 r 0.90, P < 0.01).

Similarly, there was no significant difference between patterns of richness (defined as the number of morphospecies or as the number of clusters obtained with the 631-dp dataset at a 10%, 5%, 2% and 1.2% threshold) using either the morphological or DNA barcoding approach (one-way ANOVA, F = 0.219, d.f. = 24, P = 0.925) (Fig. 6). Solenopsis richness was highest at mid-elevation (1420 m: Fig. 6). No specimens were collected at 2500 m but this could be an effect of local conditions since only a handful of other ant species were found at this site. Two Solenopsis morphospecies, both represented by a single worker (not included in the DNA analysis), were collected at 3000 m.

Discussion

Identification of Solenopsis using an integrative approach

By combining morphological and genetic analyses, we were able to group Solenopsis specimens and define units of biodiversity. Indeed, using different threshold distances to cluster DNA barcodes and delineate potential species, we were able to distinguish between well delimited clusters that were consistently grouped together and complexes of sequences showing gradual divergences. It is now recognised that no single distance threshold can be universally applied in species identification (Yassin et al. 2010). Nevertheless, the use of COI divergence among clusters may provide a preliminary indication of species richness and help to detect unexpected cryptic species (Wiemers and Fiedler 2007; Burns et al. 2008; Tänzler et al. 2012). In ants, average interspecific sequence divergences in COI generally exceed 2% (Smith et al. 2009; Wild 2009) although divergence within and among species is not consistent (Wild 2009; Jansen et al. 2009). For instance, in Linepithema, genetic divergences among species range from 0.5% to 7.8% (mean: 5.5%) while distances within species ranged from 0% to 4.6% (mean: 1.9%) (Wild 2009). It is therefore necessary to use thresholds with caution and to discuss cases of conflicts between morphology and barcoding results.

In this study, 50% of Solenopsis morphospecies (7 of 14) were well defined by clear morphological characters and were also separated from each other by relatively deep genetic divergence (5%; ‘Perfect match’ in Table 1). Contrary to this, three sympatric morphospecies (Solenopsis spp. 11, 12 and 13 – ‘Complex I’ in Table 1), easily separated on the basis of consistent morphological traits, but showed low genetic divergence (2%). For this complex, we hypothesise that three distinct species were present and that distances among them were low perhaps because of recent speciation events. It has been shown previously that closely related species can differ by only 1–3 nucleotides (Burns et al. 2007).

Four other morphospecies (Solenopsis spp. 01, 14, 15 and 21) were split into 9–15 clusters according to the available data and threshold considered (Table 1), suggesting that (1) morphological criteria used in this study for species recognition were too conservative (see also Wild 2009), (2) some genetically distinct species could not be distinguished morphologically, or (3) some species presented intraspecific divergences over 10%.

Nuclear DNA analysis rejected the third hypothesis and supported Hypotheses 1 and 2 since morphologically similar specimens were not grouped together in a monophyletic clade. Such a result has also been found in the hesperiid butterfly genus Perichares (Burns et al. 2008). Moreover, the DNA barcoding approach used here helped to detect cryptic species. For instance, some workers of Solenopsis sp. 15 were highly distinctive genetically (10%) but were very difficult to accurately distinguish on the basis of morphology (Fig. 4A, B). A reexamination of the specimens did find differences in shape of propodeum and of anteroventral petiolar process (Figs S11, S21 in Supplementary material S2) but they were subtle and easily ascribed to intraspecific variations. Fortunately, it was possible to associate gynes to workers of both clusters thanks to the DNA barcoding approach (Fig. 4C, D). It seems that gynes provide more reliable criteria than workers for separation of the two clusters (Fig. 4E, F). However, caution is needed because only 1–3 gynes were associated with confidence to each cluster, making impossible the evaluation of intraspecific variations of gyne morphology.

We admit that morphospecies sorting was probably too conservative in some cases. The fact that the number of morphospecies was almost always lower than the number of clusters based on any threshold of COI divergence (Fig. 6) suggests that lumping occurred during morphospecies identification. The reexamination of specimens allowed us to find diagnostic characters to identify two genetically well defined clusters (Figs S12, S15 in Supplementary material S2). Nevertheless, even with the help the barcoding data, it was not always straightforward to distinguish intra- from interspecific variation. For instance, specimens forming ‘Complex II’ (Table 1) were lumped together using a 10% threshold but were separated into 3–5 clusters when lower thresholds were used. This complex presented some genetic similarities (~10%) with specimens identified as S. molesta and collected from Canada and the USA (Table 1; sequences obtained from BOLD). The molesta species group sensu Creighton (1930) and Pacheco (2007) is a diverse, largely distributed complex of morphologically very similar Solenopsis species. It is possible that this complex represents a monophyletic clade but supplementary studies are needed to confirm this hypothesis. Here, Complex II corresponded to morphologically variable specimens but no clear-cut criteria were found to separate them. In addition, genetic divergence was related to elevation (Fig. 5). In the absence of evidence to the contrary so far, we consider the variation observed within Complex II to be intraspecific variability of isolated, diverging populations.

Patterns of diversity and distribution in Solenopsis

Species richness of most ant genera decreases with elevation (Lattke 2003; Dunn et al. 2010). Here, Solenopsis seems to be more abundant at mid-elevation but it is perhaps the consequence of unsuccessful DNA extractions of specimens collected at 1050 m (Supplementary material S1). On the basis of the integration of the barcoding and morphological data, numbers of Solenopsis species collected were 4, 11 and 4 species at 1050 m-C, 1420 m and 2070 m-R1, respectively. Adding non-barcoded, rare morphospecies and common species for which no barcode sequences were obtained (hypothesising that morphology alone allowed correct identification in these cases), species richness at reference sites reached 9, 13, 5, 0 and 2 species at 1050 m-C, 1420 m, 2070 m-R1, 2500 m and 3000 m, respectively. To our knowledge, records at 3000 m are the highest documented cases for the occurrence of the genus. In total, we estimated that at least 30 Solenopsis species were collected along the altitudinal gradient. Besides, similarity of Solenopsis assemblages among elevations was very low (Table 2) and most Solenopsis species (25 of 30) were found at a single altitude, indicating that species turnover and regional diversity were high. This is confirmed by the fact that sites at the same elevation and less than 4 km apart shared only a few Solenopsis species (Tables 1 and 2).

It is difficult to compare our results with published data because sampling methods and effort were different. Nevertheless, it is interesting to note that the local (α) diversity of Solenopsis found at 1050 and 1420 m are among the highest recorded. For instance, in the Otongachi forest (Ecuadorian Andes, 850 m), seven Solenopsis (morpho)species were collected with 40 pitfall traps and 40 1-m2 Winkler samples (Donoso and Ramón 2009). In Tiputini Biodiversity Station, Amazonian Ecuador (206–224 m), 15 Solenopsis (morpho)species were inventoried from deep soil layers to canopy using six sampling methods resulting in more than 200 samples (Ryder Wilkie et al. 2010). In an Amazonian forest (Brazil, 30–140 m), 900 samples were collected with three sampling methods (1-m2 Winkler samples, pitfall traps, and sardine baits: Oliveira et al. 2009), resulting in 15 Solenopsis (morpho)species. Our results suggest that the Ecuadorian Andes are a hotspot of diversity for Solenopsis ants and/or that the joint use of morphology and DNA barcoding allows better estimates of Solenopsis local diversity than morphology alone.

Inventory of Ecuadorian Solenopsis species is still at an early stage. Fernández and Sendoya (2004) found only two species, S. globularia and S. saevissima, cited from Ecuador in the literature. More recently, S. virulens (Ryder Wilkie et al. 2010) and S. cf. stricta (Donoso and Ramón 2009) were collected from the Amazonian region and the central Andes, respectively. Also, the invasive ant S. geminata was introduced to the Galápagos Archipelago and Ecuadorian mainland (Herrera and Causton 2008; Wetterer 2011), while S. gnoma, a suspected endemic of the Galápagos Islands, was recently described (Pacheco et al. 2007). It is therefore clear that most of the Solenopsis species collected in this study are potential new records for Ecuador and/or new species. Considering that 30 species is a good estimation of the Solenopsis diversity in our small study area (maximum distance between sites was 20 km), we may expect that the estimated richness at a continental scale (i.e. currently ~100 species are known in the Neotropic) is largely underestimated.

Despite the abundance and ecological importance of Solenopsis species (Ward 2000), these ants remain poorly studied because of their problematic identification. This is a major impediment for biodiversity and biogeographical studies. Our results show that morphological and DNA barcoding approaches revealed similar patterns of species richness within sites and of species turnover among sites, as observed for other ants in Madagascar (Smith et al. 2005). Montane rainforests in southern Ecuador are recognised as biodiversity hotspots for numerous taxa (Brehm et al. 2008; Strutzenberger et al. 2011). Given the high levels of species turnover among sites and of local and regional species richness of Solenopsis it seems likely that this is also the case for ants. The combined use of morphological and barcoding approaches increased the accuracy of Solenopsis identification. DNA barcoding was also helpful to associate sexual and worker castes, adding potential new diagnostic characters for species identification. In this respect, back and forth interactions between morphological and DNA barcoding approaches were facilitated by the non-invasive DNA extraction protocol.

Acknowledgements

The authors warmly thank C. Vits and B. de Roover at Copalinga Private Reserve for access to their property, J. Bendix, F. Matt, J. Zeilinger, the ‘Deutsche Forschungsgemeinschaft’ (DFG)-Research Unit 816, and the team of the ‘Estación Científica San Francisco’ for allowing and extensively facilitating their work, I. Bachy, J. Cillis, and Y. Laurent for ant digitisation, T.M. Arias-Penna and J. Peña for assistance during fieldwork. We also thank A. Austin and two anonymous referees for comments and suggestions that greatly improved the manuscript. This research was funded by the Belgian Federal Science Policy Office (BELSPO) through an Action 1 Impulse for Research and the Joint Experimental Molecular Unit (JEMU), and by the European Distributed Institute of Taxonomy (EDIT). All material has been collected under appropriate collection permits and approved ethics guidelines.

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Table 1. Delimitation of Solenopsis species by morphological and DNA barcoding approach

Fourteen Ecuadorian morphospecies, for which COI sequences were available, are represented along with Solenopsis records from GenBank and BOLD. Comparisons were made for three datasets: one with short sequences (237 bp), and two with longer sequences but including fewer samples (310 and 631 bp). Threshold values corresponded to local minima in the distributions of pairwise distances and to preset values (2% and 10%). Letters ‘a’–‘w’ refer to clusters of Ecuadorian Solenopsis. Letters ‘Ga’–‘Gp’ refer to clusters including sequences obtained from GenBank or BOLD. As a result, for each threshold and dataset, the number of different letters in the column corresponds to the number of clusters obtained with this threshold and dataset. Black cells mean that no sequence of the corresponding length was obtained. Information on elevation and collection site is given. Brief description of morphospecies and photos are provided in the Supplementary material S2 (Figs S1–S24)

Morphospecies

Clustering based on COI

Altitude (m) and Site

631 bp

310 bp

237 bp

1050

1420

2070

10.0%

5.0%

2.0%

1.2%

10.0%

9.2%

5%

3.2%

2.0%

7,0%

3,1%

Perfect match

2 (Fig. S1)

a

a

a

a

a

a

a

a

a

a

a

B,C

6 (Fig. S2)

b

b

b

b

b

b

b

b

b

b

b

C

7 (Fig. S3)

c

c

c

c

c

c

c

c

c

c

c

B

C

16 (Fig. S4)

d

d

d

d

d

d

d

d

d

d

d

B?

C

18 (Fig. S5)

e

e

e

e

e

e

e

e

e

e

e

C

22 (Fig. S6)

f

f

f

f

f

f

f

C

19 (Fig. S7)

g

g

C

Potential lumping (Complex I)

11 (Fig. S8)

h

h

h

h

h

h

h

h

h

h

h

R1,R2

13 (Fig. S9)

h

h

i

i

h

h

h

h

h

h

h

R1

12 (Fig. S10)

h

h

j

j

h

h

h

h

j

h

h

R1

Splitting

15 (Figs S11, 4A, 4C, 4E)

k

k

k

k

k

k

k

k

k

k

k

C

1 (Fig. S12)

l

l

l

l

l

l

l

l

l

l

l

R1

1 (Fig. S13)

m

m

m

m

m

m

m

m

m

m

m

B

14 (Fig. S14)

n

n

n

n

n

n

n

n

n

n

n

C

14,21 (Fig. S15)

o

o

o

o

o

o

o

o

o

o

o

C

1,15 (Fig. S16)

p

p

p

p

p

p

p

p

p

p

p

B

C

1 (Fig. S17)

q

q

q

q

q

q

q

q

q

q

q

B

C

1 (Fig. S18)

q

r

r

r

r

r

r

C

Potential splitting (Complex II)

1 (Fig. S19)

s

s

s

s

s

s

s

s

s

s

s

B,C

1,14,15 (Fig. S20)

s

t

t

t

s

s

t

t

t

s

t

C

15 (Figs S21, 4B, 4D, 4F)

s

u

u

u

s

s

t

t

u

s

u

C

1,15 (Fig. S22)

s

u

u

v

s

s

t

t

v

s

u

B,C

15 (Fig. S23)

s

u

u

w

s

s

t

t

v

s

u

C

R2

14 (Fig. S24)

s

s

t

t

v

s

u

C

Sequences from GenBank and BOLD

molesta

Ga

Ga

Ga

Ga

s

Ga

Ga

Ga

Ga

Ga

Ga

Ga

Ga

Gb

Gb

s

Ga

Ga

Gb

Gb

Ga

Gb

Ga

Gc

Gc

Gc

s

Ga

Ga

Gc

Gc

Ga

Gc

Ga

Gc

Gd

Gd

s

Ga

Ga

Gc

Gc

Ga

Gc

Ga

Gc

Gd

Ge

s

Ga

Ga

Gc

Gd

Ga

Gc

Ga

Gc

Gf

Gf

s

Ga

Ga

Gc

Gd

Ga

Gc

Ga

Gc

Gg

Gg

s

Ga

Ga

Gg

Gg

Ga

Gg

geminata

Gh

Gh

Gh

Gh

Gh

Gh

Gh

Gh

Gh

Gh

Gh

Gh

Gi

Gi

Gi

Gi

Gi

Gi

Gi

Gi

Gh

Gi

mameti

Gj

Gj

Gj

Gj

Gj

Gj

Gj

Gj

Gj

Gj

Gj

carolinensis

Gk

Gk

Gk

Gk

Gk

Gk

Gk

Gk

Gk

Gk

Gk

inv/richt

Gl

Gl

Gl

Gl

Gl

Gl

Gl

Gl

Gl

Gl

Gl

invicta

Gl

Gl

Gm

Gm

Gl

Gl

Gl

Gl

Gl

Gl

Gl

Gl

Gn

Gn

Gn

Gl

Gl

Gn

Gn

Gn

Gl

Gn

NZsp1

Go

Go

Go

Go

Go

Go

Go

Go

Go

Go

Go

MU01

Gp

Gp

Gp

Gp

Gp

Gp

Gp

Gp

Gp

Gp

Gp

No. of clusters for Ecuadorian Solenopsis

14

16

18

20

15

16

17

17

20

17

19

Total no. of clusters

21

26

33

36

22

24

26

29

33

24

31

Table 2. Faunal similarity among sites

Jaccard indices of similarity (J) between Solenopsis assemblages from five collection sites at three elevations (1050, 1420 and 2070 m above sea level). Indices were calculated for the 631-bp dataset, and included only those specimens identified on the basis of both morphology (morphospecies) and DNA barcodes

Site 1

Site 2

Morphospecies

10%

5%

2%

1.20%

1050 m-B

1050 m-C

0.40

0.29

0.38

0.38

0.38

1050 m-B

1420 m

0.30

0.36

0.31

0.31

0.20

1050 m-B

2070 m-R1

0.14

0.00

0.00

0.00

0.00

1050 m-B

2070 m-R2

0.20

0.14

0.13

0.13

0.00

1050 m-C

1420 m

0.09

0.09

0.08

0.08

0.00

1050 m-C

2070 m-R1

0.17

0.00

0.00

0.00

0.00

1050 m-C

2070 m-R2

0.00

0.25

0.20

0.20

0.00

1420 m

2070 m-R1

0.08

0.00

0.00

0.00

0.00

1420 m

2070 m-R2

0.10

0.10

0.09

0.09

0.08

2070 m-E1

2070 m-R2

0.20

0.33

0.33

0.20

0.20

Fig. 1. Neighbour-joining tree based on COI sequences (631 bp) of Solenopsis specimens obtained here (S. spp. 1–22) and from GenBank and BOLD (Accession no. and Process ID). Cluster IDs (a–w and Ga–Gp), altitude (1050, 1420 and 2070 m), geographic origin (C, Copalinga; B, Bombuscaro; R1, Reserva Biológica San Francisco-Transect T1; R2, Reserva Biológica San Francisco-NUMEX) and no. of sequences with the same haplotype when more than one (between parentheses) are given. The small tree in the right corner shows neighbour-joining tree based on wg sequences (343 bp). Same symbols are used for corresponding specimens.

Fig. 2. Proportion of pairwise genetic distances among Solenopsis haplotypes based on the (A) 237-bp, (B) 310-bp and (C) 631-bp datasets of the COI gene. Arrows indicate local minima identified for each dataset.

Fig. 3. Bayesian analysis of the concatenated COI and wg datasets. Morphospecies name, cluster IDs (a–w and Ga–Gp), altitude (1050, 1420 and 2070 m), geographic origin (C, Copalinga; R1, Reserva Biológica San Francisco-Transect T1) and Process ID in BOLD are given.

Fig. 4. Detection of cryptic species by DNA barcoding. (A, B) Some workers of Solenopsis sp. 15 presented high DNA divergence of the COI gene (>10%) but were not accurately distinguishable. (C, D) Gynes, each one placed below its associated worker, provided new criteria for species identification: longitudinal striae were restricted to the latero-basal part of propodeum in E, whereas they extended above the propodeal spiracle in F (arrows ‘a’), and an anteroventral petiolar tooth was present in E whereas absent in F (arrows ‘b’).

Fig. 5. Haplotype network of 28 COI sequences of 658 bp, from workers identified as Solenopsis spp. 01, 14 or 15 based on morphology (‘Complex II’ in Table 1). Each circle represents one haplotype and its size is related to the number of collected individuals. Length of links between circles is proportional to the number of substitutions. Morphospecies (Solenopsis spp. 01, 14 and 15) and genetic clusters (s–w) are indicated.

Fig. 6. Estimates of Solenopsis species richness at five sites spread at three altitudes and calculated on the basis of the 631-bp dataset. Here, species richness was defined as the number of Solenopsis morphospecies or as the number of clusters obtained using a 10%, 5%, 2% and 1.2% threshold.

Supplementary material S1. List of 187 Solenopsis specimens selected for DNA analyses

Sample_ID*

Process ID (BOLD)

Genus

Morphospecies

Caste

Sampling method

Sampling site

Elevation (m)

Size COI (bp)

Size wingless (bp)

33807Ssp141000QD

SOLEN024-12

Solenopsis

sp01TD

Worker

Winkler

Bombuscaro - Podocarpus National Park (1050m-B)

1050

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