diversity

Article The Megadiverse Australian : Using CO1 Barcoding to Assess Richness

Alan N. Andersen 1,*, Benjamin D. Hoffmann 2 and Kathryn Sparks 3

1 Research Institute for the Environment and Livelihoods, Charles Darwin University, Darwin, NT 0909, Australia 2 CSIRO Tropical Ecosystems Research Centre, PMB 44, Winnellie, NT 0822, Australia; [email protected] 3 South Australian Museum, North Terrace, Adelaide, SA 5000, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-457-539-513

Academic Editor: Michael Wink Received: 15 September 2016; Accepted: 3 December 2016; Published: 19 December 2016

Abstract: Melophorus is an exceptionally diverse ant genus from arid Australia that has received little taxonomic attention, such that just a fraction of its remarkable number of species is described. The Commonwealth Scientific and Industrial Research Organization’s Tropical Ecosystems Research Centre (TERC) in Darwin holds by far the most extensive collection of Melophorus, and as of September 2016 this comprised >850 sorted morphospecies. However, the reliability of such morphospecies is open to question because species delimitation is extremely challenging due to highly generalized morphology and worker polymorphism. Here we use CO1 barcoding of 401 Melophorus specimens from 188 morphospecies in the TERC collection to determine the reliability of morphologically-based species delimitations as a basis for assessing true diversity within the genus. Our CO1 data confirm the extremely challenging nature of morphologically-based species delimitation within Melophorus, and suggest substantially higher diversity than that indicated by morphospecies. We found many cases where combinations of high (>10%) CO1 divergence, polyphyly, sympatric association, and morphological differentiation indicated that single morphospecies represented multiple lineages. Overall, our analysis indicates that the 188 morphospecies barcoded represent at least 225 independent CO1 lineages. We discuss these results in terms of both their limitations and implications for estimating the total number of species in this exceptionally diverse, arid-adapted ant genus.

Keywords: ant diversity; biological species; morphospecies; species delimitation; sympatric association

1. Introduction Melophorus Lubbock 1883 is an exceptionally diverse ant genus endemic to Australia, occurring primarily in arid and semi-arid regions. Despite it being a dominant component of inland ant faunas throughout the continent [1,2], it has attracted little research attention from either ecologists or taxonomists. Ecological studies have focussed on a single species, the “honeypot” ant M. bagoti Lubbock, 1883 [3], documenting its extreme thermophilia [4,5] and other behavioural aspects [6–9]. A small number of other species have also been subject to behavioural studies [10–12]. As highly thermophilic, polymorphic formicines, many species of Melophorus are strongly convergent both morphologically and behaviourally with in North American deserts [13] and in northern Africa, southern Europe, and the Middle East [14]. However, the generally conservative morphology of Melophorus belies its extremely diverse biology. Although most species are generalist predators and scavengers, the genus includes specialist predators of ant brood (from the M. fulvihirtus Clark, 1941 and M. anderseni Agosti, 1997 species groups [15,16]) and

Diversity 2016, 8, 30; doi:10.3390/d8040030 www.mdpi.com/journal/diversity Diversity 2016, 8, 30 2 of 13 specialist granivores (species of the M. wheeleri Forel, 1910 species group), an extremely unusual condition in formicines [17]. Only about 30 species of Melophorus have been described (Shattuck 1999), almost all prior to 1950 and involving many different authors, but this represents just a small fraction of even the known fauna [18]. For example, 45 Melophorus species were recorded from near Mt Isa in northwesten Queensland [19], 28 species from Purnululu National Park in northern Western Australia [20], 30 species from Uluru National Park in Central Australia [21], and 41 species from the Great Western Woodlands of southern Western Australia [22], with little overlap among these local faunas. It has recently been suggested that Melophorus is likely to contain well over 1000 species, based on holdings in the Commonwealth Scientific and Industrial Research Organization’s Tropical Ecosystems Research Centre (TERC) in Darwin where the >10,000 pinned Melophorus specimens have been sorted into >850 morphospecies [23]. This would make Melophorus one of the world’s most-species rich ant genera, and by far the richest with such a limited distribution. However, the generalized morphology of most Melophorus species makes species delimitation extremely challenging, and so the reliability of the morphospecies sorting in the TERC collection requires validation. Integrated morphological, behavioural, and genetic analyses have consistently validated the sorting of morphospecies in the TERC collection from other highly diverse and taxonomically challenging Australian genera such as Monomorium [24–26] and Iridomyrmex [27]. Indeed, CO1 data from specimens from the M. aeneovirens group suggest that the sorting of Melophorus morphospecies in the TERC collection is in fact highly conservative [23]. In this paper, we use CO1 barcoding of hundreds of Melophorus specimens representing almost all known species groups to assess the reliability of morphologically based species delimitations in the TERC collection, as a basis for gauging true diversity within the genus. Ideally, other genes would also be included in addition to CO1 in order to provide more-definitive results. However, there is a trade-off between number of genes and number of samples that can be feasibly analyzed, and we wished to maximize the number of samples. We also provide images of representatives of all common species groups in order to illustrate morphological diversity within the genus.

2. Materials and Methods In 2015, 401 specimens representing 188 morphospecies from the TERC collection (Table S1) were sequenced for the CO1-barcoding region on the basis of DNA extraction and sequencing from either legs (larger species) or whole specimens (smaller species) through the BOLD Barcode of Life Data System (see [28] for extraction details). These specimens represent nearly all known species groups of Melophorus. Specimens of the melophorine genera Notoncus Emery, 1895 (single species) and Prolasius Forel, 1892 (13 species), along with a specimen of Teratomyrmex greavesii McAreavey, 1957, which the senior author suspected belongs to Prolasius, were similarly barcoded for use as outgroups (Table S2). A CO1 sequence of a species of the melophorine genus Myrmecorhyncus obtained from Genbank (DQ353336) was also incorporated into the analysis. The DNA sequences were trimmed to 612 bases using Bioedit 7.0.9 [29] and translated into proteins using MEGA v.5 [30] to check for the presence of nuclear paralogues. PartitionFinder V1.1.1 [31] was used to determine the best evolutionary model partitioning scheme for codon positions 1, 2, and 3 using the Bayesian Information Criterion (BIC) and the “greedy” algorithm. Bayesian analysis was performed for 20 million generations, sampling every 1000 generations using MrBayes (3.1.2) through the CIPRES Science Gateway V. 3.1 [32]. Stationarity was assumed to have been reached once the standard deviation of split frequencies fell below 0.01. Additionally, TRACER 1.4 [33] was used to check for chain convergence and the first 25% of trees were discarded as burn-in. Genetic Distances were calculated in MEGA v.5 using the Kimura-2 parameter model. There is no arbitrary level of CO1 divergence that can be used to define species; however, the level of variation within an ant species is typically 1%–3% [34], although it can be substantially higher [35]. We based our assessments of species delimitation on a combination of genetic divergence, morphological differentiation, phylogenetic structure, and sympatric association. Diversity 2016, 8, 30 3 of 13

Diversity 2016, 8, 30 3 of 12 3. Results 3. Results Our CO1 tree shows all Melophorus specimens forming a well-supported (PP = 0.95) clade Our CO1 tree shows all Melophorus specimens forming a well-supported (PP = 0.95) clade (Figure (Figure S1). Teratomyrmex greavesii is embedded within the Prolasius clade. Within Melophorus, the CO1 S1). Teratomyrmex greavesii is embedded within the Prolasius clade. Within Melophorus, the CO1 tree tree shows a primary division (PP = 1) between species from what is described as the M. aeneovirens shows a primary division (PP = 1) between species from what is described as the M. aeneovirens radiation by [18], including the M. bagoti group (Figure1a,b), the M. froggatti group (Figure1c), radiation by [18], including the M. bagoti group (Figure 1a,b), the M. froggatti group (Figure 1c), Group Group A (Figure1d), Group O (Figure1e) and the M. aeneovirens gp. (Figure1f), and all other species A (Figure 1d), Group O (Figure 1e) and the M. aeneovirens gp. (Figure 1f), and all other species (Figure (Figure2). This second branch of the primary division, containing all species groups outside the 2). This second branch of the primary division, containing all species groups outside the M. M. aeneovirens radiation, is very poorly resolved with low branch support at the base (PP = 0.59). aeneovirens radiation, is very poorly resolved with low branch support at the base (PP = 0.59). However, specimens from the M. potteri group form a well-defined clade (PP = 1) within it, and However, specimens from the M. potteri group form a well-defined clade (PP = 1) within it, and the the two complexes recognized by [18] are recovered as well-supported (PP = 1) sub-clades. One of two complexes recognized by [18] are recovered as well-supported (PP = 1) sub-clades. One of these these is characterised by a projecting clypeus and dentate mandibles (specimens 490, 721 and 723; is characterised by a projecting clypeus and dentate mandibles (specimens 490, 721 and 723; complex complex 1, Figure3a), and in the other the clypeus is not projecting and the mandibles are massive and 1, Figure 3a), and in the other the clypeus is not projecting and the mandibles are massive and edentateedentate exceptexcept forfor aa largelarge apicalapical toothtooth (specimens(specimens 06 0644 and 722; complex complex 2, 2, Figu Figurere 3b).3b). In In both both these these complexescomplexes thethe mandibularmandibular andand maxillarymaxillary palps are highly vestigial. vestigial. There There is is a a third third complex complex within within thethe group group that that was was not not sequenced, sequenced, known known from from two tw morphospecieso morphospecies from from the the central central and and southern southern arid zones.arid zones. These These have ahave typical a typical mesosoma mesosoma for the forM. the potteri M. potterigroup, group, and the and head the similarlyhead similarly lacks dorsallacks setae,dorsal but setae, neither but neither the clypeus the clyp noreus mandibles nor mandibles are modified, are modified, and the and palps the are palps relatively are relatively short but short not vestigialbut not vestigial (complex (complex 3, Figure 33,c). Figure Interestingly, 3c). Interestingly, the M. potteri the M.group potteri is showngroup asis shown containing as containing an additional an cladeadditional (complex clade 4; (complex specimens 4; 066—068), specimens consisting 066—068), of consisting a recently of discovered a recently species discovered from species southwestern from Westernsouthwestern Australia Western known Australia only from known reproductives only from that reproductives have a unique, that horseshoe-shaped have a unique, petiolar horseshoe- node (Figureshaped3 d).petiolar An absence node (Figure of workers 3d). An and absence the peculiar of workers morphology and the of peculiar the female morphology (Figure3d) of suggest the female that this(Figure species 3d) is suggest a social that parasite. this species The CO1 is treea soci recoveredal parasite. none The of theCO1M. tree fulvihirtus recovered(Figure none4), ofM. the ludius M. (Figurefulvihirtus5), M.(Figure wheeleri 4), (FigureM. ludius6) or(FigureM. fieldi 5), M.(Figure wheeleri7) radiations (Figure 6)of or [ M.18], fieldi or many (Figure of their7) radiations component of species[18], orgroups many of (Figure their compon2). ent species groups (Figure 2).

(a) (b)

(c) (d)

Figure 1. Cont.

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(e) (f) (e) (f) Figure 1. Representatives of the M. aeneovirens radiation. (a) M. iridescens; (b) M. bagoti; (c) member of FigureFigure 1. 1.Representatives Representatives of of the theM. M. aeneovirens aeneovirensradiation. radiation.( (aa))M. M. iridescensiridescens;(; (bb)) M.M. bagoti;(; (c) member of the M. froggatti group; (d) member of Group A; (e) member of Group O; (f) member of the M. thetheM. M. froggatti froggattigroup; group; (d) member(d) member of Group of Group A; (e) memberA; (e) member of Group of O;Group (f) member O; (f) ofmember the M. aeneovirensof the M. aeneovirens group. All specimens are minor workers. group.aeneovirens All specimens group. All are specimens minor workers. are minor workers.

Figure 2. Bayesian 50 percent majority rule summary tree. The tree shows the overall clade structure Figure 2. Bayesian 50 percent majority rule summary tree. The tree shows the overall clade structure Figurefor the 2. Bayesian 401 sequenced 50 percent specimens majority ruleof Melophorus summary tree.. Nodes The treewith shows ≥95 theand overall ≥70 Bayesian clade structure posterior for for the 401 sequenced specimens of Melophorus. Nodes≥ with≥ ≥95 and ≥70 Bayesian posterior theprobabilities 401 sequenced are specimensindicated by of Melophorusblack and .red Nodes circles, with respectively.95 and 70The Bayesian full tree posterior is provided probabilities in Figure probabilities are indicated by black and red circles, respectively. The full tree is provided in Figure areS1. indicated by black and red circles, respectively. The full tree is provided in Figure S1. S1.

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(a) (b) (a) (b)

(c) (d) (c) (d) Figure 3. Representatives of the M. potteri group. (a) Member of complex 1; (b) member of complex 2; Figure 3. Representatives of the M. potteri group. (a) Member of complex 1; (b) member of complex 2; Figure(c) member 3. Representatives of complex 3; ofand the (d M.) member potteri group. of complex (a) Member 4. of complex 1; (b) member of complex 2; (c) member of complex 3; and (d) member of complex 4. (c) member of complex 3; and (d) member of complex 4.

(a) (b) (a) (b)

(c) (d) (c) (d) Figure 4. Representatives of the M. fulvihirtus radiation. (a) Member of the M. Group I; (b) M. FigureFigureanderseni 4. Representatives4.; (Representativesc) M. fulvihirtus of; theandof theM. (d ) fulvihirtusM. member fulvihirtus ofradiation. Melophorus radiation. (a) Group Member(a) Member G. of the ofM the. Group M. Group I; (b) M.I; ( andersenib) M. ; (c)anderseniM. fulvihirtus; (c) M.; andfulvihirtus (d) member; and (d of) memberMelophorus of MelophorusGroup G. Group G.

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(a) (b) (a) (b)

(c) (d) (c) (d) Figure 5. Representatives of the M. ludius radiation. (a) M. ludius; (b) M. mjobergi; (c) M. hirsutus; and Figure 5. 5. RepresentativesRepresentatives of of the the M.M. ludius ludius radiation.radiation. (a) (M.a) M.ludius ludius; (b);( M.b) mjobergiM. mjobergi; (c) ;(M.c )hirsutusM. hirsutus; and; (d) member of Melophorus Group D. (andd) member (d) member of Melophorus of Melophorus GroupGroup D. D.

(a) (b) (a) (b) Figure 6. Representatives of the M. wheeleri radiation. (a) Member of Melophorus Group H; (b) Member Figure 6. Representatives of the M. wheeleri radiation. (a) Member of Melophorus Group H; (b) Member Figureof the M. 6. wheeleriRepresentatives group. of the M. wheeleri radiation. (a) Member of Melophorus Group H; (b) Member of the M. wheeleri group.

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(a) (b)(c)

(d) (e)(f)

(g) (h)(i)

Figure 7. Representatives of the M. fieldi radiation. (a) Member of the M. perthensis group; (b) member Figure 7. Representatives of the M. fieldi radiation. (a) Member of the M. perthensis group; (b) member of Melophorusof MelophorusGroup Group J; (c )J; member (c) member of the ofM. the fieldi M. group;fieldi group; (d) member (d) member of the M. of turnerithe M.group; turneri ( egroup;) member (e) ofmember the M. of bruneus the M. bruneusgroup; group; (f) member (f) member of the ofM. the pillipes M. pillipesgroup; group; (g) member (g) member of Melophorus of MelophorusGroup Group E; (E;h) ( memberh) member of Melophorusof MelophorusGroup Group M; M; and and (i) member(i) member of Melophorusof MelophorusGroup Group N. N.

The CO1 tree revealed six cases where specimens sorted as two morphospecies are likely to be The CO1 tree revealed six cases where specimens sorted as two morphospecies are likely to be conspecific: sp. O (specimen 195) and GWW sp. Y (889 and 890) within the M. turneri gp.; GWW sp. conspecific: sp. O (specimen 195) and GWW sp. Y (889 and 890) within the M. turneri gp.; GWW sp. CE (949) and GWW sp. BC (257) within the M. fieldi gp.; GWW sp. C (256) and sp. Eurardy 25 (259) CE (949) and GWW sp. BC (257) within the M. fieldi gp.; GWW sp. C (256) and sp. Eurardy 25 (259) within the M. fieldi gp.; NATT sp. AN (573) and NATT sp. AF (572) within Group A; sp. P (612) and within the M. fieldi gp.; NATT sp. AN (573) and NATT sp. AF (572) within Group A; sp. P (612) and sp. TREND AO (615) within Group B; and Eurardy sp. BB (074, 075) and Eurardy sp. M (076 and 077) sp. TREND AO (615) within Group B; and Eurardy sp. BB (074, 075) and Eurardy sp. M (076 and 077) within Group A (Figure S1). However, there were many more cases where a morphospecies was within Group A (Figure S1). However, there were many more cases where a morphospecies was resolved as polyphyletic with high (>10%) genetic divergence, and often in sympatry. resolved as polyphyletic with high (>10%) genetic divergence, and often in sympatry. The apparent occurrence of multiple independent lineages within a morphospecies was The apparent occurrence of multiple independent lineages within a morphospecies was particularly prevalent in the M. fieldi group, which has especially conservative morphology. One particularly prevalent in the M. fieldi group, which has especially conservative morphology. example (Melophorus sp. 30) is a very gracile, glabrous, pale yellow morphospecies with a two-toned One example (Melophorus sp. 30) is a very gracile, glabrous, pale yellow morphospecies with a (paler anteriorly) head that occurs throughout arid and semi-arid Australia (Figure 6c). The CO1 data two-toned (paler anteriorly) head that occurs throughout arid and semi-arid Australia (Figure6c). show that mean genetic distance among sequences in this morphospecies is 8.4% with a maximum The CO1 data show that mean genetic distance among sequences in this morphospecies is 8.4% with a distance of 18.8%, and indicate that it is highly polyphyletic; the nine specimens analysed are shown maximum distance of 18.8%, and indicate that it is highly polyphyletic; the nine specimens analysed as representing six lineages (sp. 30 A–F in Figure 8), four of which occur at a single locality (Eurardy are shown as representing six lineages (sp. 30 A–F in Figure8), four of which occur at a single locality Station, WA; shown as sw WA in Figure 8). Morphological variation among the taxa is evident upon (Eurardy Station, WA; shown as sw WA in Figure8). Morphological variation among the taxa is close re-inspection. For example, among the four species co-occurring at Eurardy Station, in one (sp. evident upon close re-inspection. For example, among the four species co-occurring at Eurardy Station, 30-A) the posterior face of the petiolar node is concave rather than straight in profile, another (sp. 30- in one (sp. 30-A) the posterior face of the petiolar node is concave rather than straight in profile, D) has larger eyes, and another (sp. 30-E) has smaller overall body size. Another example is another (sp. 30-D) has larger eyes, and another (sp. 30-E) has smaller overall body size. Another Melophorus sp. 82, a sparsely hairy, brownish morphospecies that also occurs throughout arid and example is Melophorus sp. 82, a sparsely hairy, brownish morphospecies that also occurs throughout semi-arid Australia; the CO1 data show the 11 analyzed specimens as occurring in at least six arid and semi-arid Australia; the CO1 data show the 11 analyzed specimens as occurring in at least six polyphyletic lineages (sp. 82 A–F in Figure 8). Again, careful re-inspection of the specimens reveals previously undetected morphological variation relating to pilosity, relative scape length, and head shape. Melophorus sp. 11, occurring throughout the Top End of the Northern Territory, is shown as

Diversity 2016, 8, 30 8 of 13 polyphyletic lineages (sp. 82 A–F in Figure8). Again, careful re-inspection of the specimens reveals previouslyDiversity 2016, undetected 8, 30 morphological variation relating to pilosity, relative scape length, and head8 of 12 shape. Melophorus sp. 11, occurring throughout the Top End of the Northern Territory, is shown as representingrepresenting atat leastleast fourfour lineageslineages fromfrom fourfour separateseparate cladesclades (sp.(sp. 11 A–DA–D inin FigureFigure8 8).). OnceOnce again,again, closeclose re-inspectionre-inspection of of the the specimens specimens reveals reveals a range a range of previously of previously undetected undetected morphological morphological variation amongvariation the among taxa. the taxa.

FigureFigure 8.8. ExtractExtract from from the the full full CO1 CO1 tree tree (Figure (Figure S1)—I. S1)—I. The treeThe indicatestree indica thattes sp. that 11 (red),sp. 11 sp. (red), 30 (green), sp. 30 and(green), sp.82 and (blue) sp. from82 (blue) the M. from fieldi thegroup M. eachfieldi representgroup each several represent species, severa as indicatedl species, by as different indicated letters. by NTdifferent = Northern letters. Territory; NT = Northern SA = South Territory; Australia; SA = South WA = Australia; Western Australia. WA = Western Australia.

SpeciesSpecies ofof thetheM. M. ludius ludiusgroup group are are the the smallest smallest (ca. (ca. 1.5 1.5 mm mm total total length) length) within within the genus,the genus, and alsoand havealso have very highlyvery highly generalized generalized (plagiolepidine-like) (plagiolepidine-like) morphology morphology (Figure5 a);(Figure it is therefore 5a); it is not therefore surprising not thatsurprising there werethat severalthere were cases several where CO1cases datawhere indicated CO1 data multiple indicated taxa withinmultiple a morphospecies.taxa within a Formorphospecies. example, specimens For example, identified specimens as Melophorus identifiedsp. as AJ Melophorus from Eurardy sp. StationAJ from in Eurardy Western Station Australia in occurWestern in threeAustralia clades occur at that in three locality, clades showing at that a locality, mean K2P showing distance a mean of 11% K2P (Figure distance9). Morphometric of 11% (Figure variation9). Morphometric among the variation taxa, especially among the relating taxa, especially to headshape, relating is to evident head shape, upon re-inspectionis evident upon of there- specimens.inspection of Specimens the specimens. from Specimens the related from Group the Drelated sorted Group as a singleD sorted morphospecies as a single morphospecies occurring in highoccurring rainfall in high areas rainfall of northwestern areas of northwestern Australia are Au indicatedstralia are as indicated representing as representing four species four (Figure species 10). Melophorus(Figure 10). bagoti Melophorusshows verybagoti high shows (up tovery 21%) high CO1 (up variation to 21%) that CO1 is notvariation related that to proximity is not related between to samplesproximity and between is therefore samples suggestive and is therefore of a species suggestive complex of (Figure a species S1). complex Overall, (Figure the CO1 S1). data Overall, from thethe 188CO1 barcoded data from morphospecies the 188 barcoded are indicatedmorphospecies as representing are indicated at least as representing 225 species. at least 225 species.

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Figure 9. Extract from the full CO1 tree (Figure S1)—II. The clade represents species from the M. ludius FigureFigure 9. Extract 9. Extract from from the the full full CO1 CO1 tree tree (Figure (Figure S1)—II. S1)—II. TheThe cladeclade represents represents species species from from the M.the ludius M. ludius group with reddish head and mesosoma and contrasting black gaster. Specimens sorted as Melophorus groupgroup with with reddish reddish head head and and mesosoma mesosoma and and contrasting contrasting blackblack gaster. gaster. Specimens Specimens sorted sorted as Melophorus as Melophorus Eurardy sp. AJ (ludius gp.) appear to represent three species (A–C), all from Eurardy Station in EurardyEurardy sp. sp.AJ AJ(ludius (ludius gp.)gp.) appearappear to to represent represent three three species specie (A–C),s all(A–C), from Eurardyall from Station Eurardy in Western Station in Western Australia. SA = South Australia; WA = Western Australia. WesternAustralia. Australia. SA = SouthSA = South Australia; Australia; WA = Western WA = Western Australia. Australia.

FigureFigure 10. Extract 10. Extract from from the the full full CO1 CO1 tree tree (Figure (Figure S1)—II S1)—III.I. TheThe clade clade represents represents species species from from Group Group D D Figure 10. Extract from the full CO1 tree (Figure S1)—III. The clade represents species from Group D occurringoccurring in northwestern in northwestern Au Australia.stralia. Specimens Specimens sorted asasMelophorus Melophorussp. sp. 10 10 (Group (Group D) D) likely likely represent represent occurringfour species in northwestern (A–D). NT =Au Northernstralia. Territory;Specimens WA sorted = Western as Melophorus Australia. sp. 10 (Group D) likely represent four species (A–D). NT = Northern Territory; WA = Western Australia. four species (A–D). NT = Northern Territory; WA = Western Australia. 4. Discussion 4. Discussion 4. Discussion The CO1 gene is used primarily to inform species boundaries and relationships among closely The CO1 gene is used primarily to inform species boundaries and relationships among closely relatedThe CO1 species gene [36 is], used and has primarily limited reliabilityto inform for spec informingies boundaries deeper phylogenetic and relationships relationships among [37, 38closely]. related species [36], and has limited reliability for informing deeper phylogenetic relationships relatedThe species deeper clade [36], structure and has of ourlimited CO1 tree,reliability which showed for informing generally poordeeper recovery phylogenetic of the species relationships groups [37,38]. The deeper clade structure of our CO1 tree, which showed generally poor recovery of the [37,38].recognised The deeper by [18 clade], is therefore structur note of reliable. our CO1 However, tree, which our CO1 showed tree did generally recover thepoorM. recovery aeneovirens of the speciesradiation groups of recognised [18]. It also recoveredby [18], is the therefore highly distinctive not reliable.M. potteri However,group, our and CO1 indicated tree thatdid arecover newly the species groups recognised by [18], is therefore not reliable. However, our CO1 tree did recover the M. aeneovirensdiscovered, radiation apparently of [18]. parasitic It also species recovered belongs the to highly it, despite distinctive having M. very potteri different group, morphology. and indicated M. aeneovirens radiation of [18]. It also recovered the highly distinctive M. potteri group, and indicated that Givena newly that discovered, ant social parasites apparently and their parasitic hosts arespec typicallyies belongs closely to related, it, despite we assume having that very this different new that a newly discovered, apparently parasitic species belongs to it, despite having very different morphology.species is aGiven parasite that of ant other social members parasites of the andM. potteri their group.hosts are typically closely related, we assume morphology. Given that ant social parasites and their hosts are typically closely related, we assume that this newOur CO1species data is a strongly parasite support of other the members genetic differentiationof the M. potteri of group. our morphospecies, showing that this new species is a parasite of other members of the M. potteri group. thatOur justCO1 a verydata smallstrongly number support of the the 188 genetic morphospecies differentiation analysed of areour likely morphospecies, to be conspecific showing with that otherOur CO1 morphospecies. data strongly Such support “splitting” the appearedgenetic differe to be farntiation less of of an our issue morphospecies, than apparent “lumping”, showing that just a very small number of the 188 morphospecies analysed are likely to be conspecific with other just awith very our small CO1 number analysis indicatingof the 188 many morphospecies examples of analysed multiple lineages are likely within to be single conspecific morphospecies. with other morphospecies. Such “splitting” appeared to be far less of an issue than apparent “lumping”, with morphospecies.CO1 data are Such generally “splitting” reliable appeared in informing to be species far less boundaries of an issue [36 than], and apparent commonly “lumping”, reveal that with our CO1 analysis indicating many examples of multiple lineages within single morphospecies. CO1 our CO1species analysis previously indicating considered many variable examples and widespread of multiple are lineages likely to within represent single multiple morphospecies. species [39,40]. CO1 data are generally reliable in informing species boundaries [36], and commonly reveal that species data Inare some generally cases, CO1reliable data in can informing provide misleading species boundaries results due [36], to Wolbachia and commonly-associated reveal divergence that species in previouslythe mitochondrial considered genome, variable incomplete and widespread lineage sorting, are likely or introgressive to represent hybridisation multiple species [41,42 ].[39,40]. CO1 In previously considered variable and widespread are likely to represent multiple species [39,40]. In someanalysis cases, CO1 has sometimes data can provide provided misleading conflicting dataresults in studies due to of Wolbachia ant genera,-associated such as Cataglyphis divergence[43 in]. the some cases, CO1 data can provide misleading results due to Wolbachia-associated divergence in the mitochondrial genome, incomplete lineage sorting, or introgressive hybridisation [41,42]. CO1 mitochondrial genome, incomplete lineage sorting, or introgressive hybridisation [41,42]. CO1 analysis has sometimes provided conflicting data in studies of ant genera, such as Cataglyphis [43]. analysis has sometimes provided conflicting data in studies of ant genera, such as Cataglyphis [43]. However, we are unaware of any study showing a single ant species to include multiple sympatric However, we are unaware of any study showing a single ant species to include multiple sympatric and morphologically differentiated clades with very high CO1 divergence, as was often the case in and morphologically differentiated clades with very high CO1 divergence, as was often the case in our study for TERC morphospecies. our study for TERC morphospecies.

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However, we are unaware of any study showing a single ant species to include multiple sympatric and morphologically differentiated clades with very high CO1 divergence, as was often the case in our study for TERC morphospecies. Analysis of nuclear markers is needed to provide more definitive results, especially where the results from mitochondrial DNA are in conflict with the morphology. However, the widespread pattern of high CO1 divergence, polyphyly, sympatric association, and morphological differentiation within morphospecies as sorted in the TERC collection make it most likely that such sorting has been conservative. It confirms the extremely challenging nature of morphologically-based species delimitation within Melophorus, which is to be expected when extreme diversity combines with highly generalized morphology and worker polymorphism. Useful morphometric characters such as relative scape length and head width show allometric variation within a species, and so without full nest series it can be highly problematic to determine whether or not similar taxa from separate collections are conspecific. This is especially the case when assessing specimens with slightly different morphology collected from different locations, where species differentiation needs to be disentangled from both allometric and geographic variation. Such issues have been well-documented in other polymorphic ant genera. For example, in many cases species delimitation is virtually impossible in the absence of major workers in the exceptionally diverse genus Pheidole [44]. Even in genera with complex morphology, such as Atta, it is often not possible to differentiate species based on minor workers, despite the species being highly divergent phylogenetically [45]. The TERC collection holds >850 Melophorus morphospecies. Our CO1 data indicate that the number of actual species in the collection is substantially higher. This is consistent with results from CO1 analyses of specimens of Monomorium from the TERC collection [23,24,26], as well as analyses of CO1 data from >1000 sequenced specimens from the TERC collection belonging to other diverse genera such as Iridomyrmex, Camponotus, Rhytidoponera, and Tetramorium (A. Andersen, unpublished data). Given that vast areas of inland Australia have never been surveyed for , we would not be surprised if the total number of Melophorus species is more than 1500. This is truly remarkable diversity for an arid-adapted ant genus confined to a single biogeographic realm. The number of Melophorus species is at least twenty times higher than the world’s next richest specialist arid-adapted ant genus (Cataglyphis). It is also an order of magnitude higher than that for any other ant genus with such a biogeographically limited range. Indeed, Melophorus should be viewed as being among the top few of the world’s richest ant genera, along with the cosmopolitan Camponotus and Pheidole that are found in most of the world’s terrestrial habitats that support ants. Many other ant genera are also exceptionally diverse in arid Australia, where Monomorium, Camponotus, Pheidole, Tetramorium, Meranolus, and Rhytidoponera are also all represented by hundreds of species [18,46]. Such remarkable diversity has been attributed to Australia’s unique history of aridity and associated patterns of speciation and extinction during the Pleistocene glaciations, when massive movement of sand likely resulted in a highly fragmented biota confined to a very large number of isolated refugia [46].

Supplementary Materials: The following are available online at www.mdpi.com/1424-2818/8/4/30/s1. Figure S1: Fifty percent majority rule Bayesian consensus tree of Melophorus. Black circles indicate posterior probability (PP) > 0.95, green circles indicate PP > 0.7 < 0.95 and red circles indicate PP > 0.5< 0.7. Table S1: List of specimens of Melophorus that were CO1-barcoded for this study. Table S2: List of outgroup specimens that were CO1-barcoded for this study. Acknowledgments: We thank our many colleagues who have assisted with the ant surveys that have contributed Melophorus specimens to the TERC collection over many years, especially Lyn Lowe, Tony Hertog, Magen Pettit, and Jodie Hayward, and more recently Gabriela Arcoverde, Adam Cross, Sarah Bonney, and Stefanie Oberprieler for specimens used for CO1 analysis. We are grateful to Jodie Hayward, Alexandra Gutierrez, and Stefanie Oberprieler for preparing the images in Figures1 and3–7, to Jodie Hayward and Stefanie Oberprieler for assisting with the preparation of specimens for CO1 analysis, and to Barry Bolton, Phil Ward, and Andy Austin for their comments on the draft manuscript. Diversity 2016, 8, 30 11 of 13

Author Contributions: A.N.A. conceived the study, developed the TERC collection, and wrote the first draft of the manuscript; B.D.H. helped develop the TERC collection, and contributed to the writing of the paper; K.S. undertook the analysis of the CO1 data, and contributed to the writing of the paper. Conflicts of Interest: The authors declare no conflict of interest.

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