Phylogenetic analysis and systematic position of two new species of the ant genus Crematogaster (Hymenoptera, Formicidae) from Southeast Asia

Two distinct new species of the ant genus Crematogaster, C. khmerensis sp. nov. and C. pfeifferi sp. nov., are described from Cambodia and Malaysia, respectively. The two species are unique among Asian Crematogaster in that they have vertically directed propodeal spines, but their systematic positions have not been determined based on morphological characters alone. Molecular phylogenetic analysis of 89 Crematogaster taxon matrices previously published plus C. khmerensis sp. nov., using nuclear genes, reveals that C. khmerensis sp. nov. is nested within the Australo-Asian Crematogaster clade. Morphological assignment of the developed pronotal shoulders implies a close relationship between C. khmerensis sp. nov. and the C. tetracantha-group. Based on molecular and morphological evidence, we erect a new species group, C. khmerensis-group, to contain C. khmerensis sp. nov. and C. pfeifferi sp. nov. Divergence time estimates using MCMCTree shows that the root node of the C. khmerensis sp. nov. terminal is estimated to be of middle Miocene age at 15 million years old. The position of the C. khmerensis-group well supports the Orientalto Australian-region dispersal history that has been proposed for the Australo-Asian Crematogaster clade.


Introduction
Ants (Formicidae) are one of the most familiar insect groups and they play important ecological roles in the ecosystems they inhabit (Hölldobler & Wilson 1990).They are abundant in terms of biomass, are distributed worldwide and form a diverse family, particularly in the tropics.Approximately 13 000 ant species have been described to date (Bolton 2014), and it is estimated that this number will increase to approximately 20 000 species in future (Hölldobler & Wilson 1990).
Ant taxonomy has been extensively investigated in recent years, with most taxonomic treatments typically presented as regional or systematic revisions.However, species with unique morphological characters are occasionally described separately from those comprehensive revisions.For example the Carebara phragmotica species clade and Tetraponera phragmotica have plug-shaped heads in the major worker caste (Ward 2006;Fischer et al. 2015); Crematogaster masukoi and C. myops have small compound eyes consisting of 5-6 ommatidia in the worker caste (Hosoishi et al. 2010).The possession of a phragmotic head suggests that the head is used to plug the nest entrance, and reduced compound eyes suggest a subterranean mode of life.Such derived states, as inferred by comparison with congenerics, are probably adaptations for particular life histories (e.g., predatory behavior, nesting habitat selection).Consequently, studies of distinct morphological characteristics are important contributions to ant taxonomy.While these distinct morphological forms (autapomorphies) can easily be distinguished from other more typical forms, and can therefore be described as new taxa, it can be difficult to determine their phylogenetic position.Thus, while uniqueness makes it easy to distinguish one taxon from others, elucidating the systematics of such taxa can be difficult.
Recent molecular analyses have revealed that some traditional taxa are not monophyletic e.g., (Cerapachyinae in Moreau et al. 2006, subgenera Paracrema and Physocrema in Blaimer 2012c, Aphaenogaster and Tetramorium by Ward et al. 2015).Indeed, since molecular data can resolve uncertainties in classification based on morphological data, an integrated approach that uses morphological data in conjunction with molecular phylogenetic analysis should be undertaken if fresh material is available.
The genus Crematogaster (Myrmicinae: Crematogastrini) is one of the hyperdiverse groups of ants, containing more than 400 species (Blaimer 2012b).The taxonomy has typically been examined as part of regional or systematic revisions (Longino 2003;Hosoishi & Ogata 2009;Blaimer & Fisher 2013), but some Crematogaster taxa with unique features have been recognized; for example, the C. borneensisgroup has reduced 10-segmented antennae and has mutualistic relationships with the ant-plant genus Macaranga (Feldhaar et al. 2016), several species in the subgenus Orthocrema have reduced compound eyes (Hosoishi et al. 2010), C. paradoxa and related species have distinctly elongated propodeal spines (Creighton 1945), C. reticulata has a strongly reticulated body surface (Hosoishi 2009), and the queen of C. cylindriceps has a phragmotic head (Wheeler 1927;Yamane et al. 2011).The systematic positions of these taxa are usually proposed based on molecular or morphological data (Blaimer 2012b;Hosoishi & Ogata 2016), but the taxonomic affiliations are not always clear in some species.
In the course of a recent examination of Crematogaster specimens collected from Southeast Asia, we found two distinct species that appear to be new to science.None of the authors have ever seen any morphologically related species in a museum collection before.While the new species clearly belong to the subgenus Crematogaster sensu stricto (Blaimer 2012b), assignment to any of the species groups was not possible.The two species are considered to be distinct in that they both have vertically directed propodeal spines, which clearly distinguishes them from other Asian species of Crematogaster; however, their systematic positions are unknown.
The aims of this study were therefore to (1) clarify the phylogenetic position of these distinct Crematogaster ants using molecular phylogenetic analysis; (2) describe the two taxa as new species; (3) present the systematics of the two new species using morphological characters and molecular phylogenetic analysis.Morphological data in conjunction with molecular phylogenetic analysis will provide more comprehensive systematics of the two 'unique' species.

Sources of material
Type and non-type specimens were examined and/or deposited in the collections listed below.The species of Crematogaster from the Australian region are reviewed based on type and non-type specimen images available on AntWeb (https://www.antweb.org).Codes for public institutions generally follow those in Brandão (2000).

Observation
Most observations were made on a Leica M205C stereo microscope.Images were taken using a Canon EOS 50D with a Canon MP-E 65 mm 1-5 × Macro lens, then processed using Combine ZM.

Measurements and indices
Measurements were made under a Leica M205C stereo microscope using ocular micrometers.All measurements are expressed in millimeters, recorded to the second decimal place.The measurements for petiole and postpetiole follow Longino (2003).Reactions were carried out at 10 µl volumes in a PCR Thermal Cycler MP (TaKaRa Bio Inc.) under the following conditions: first 40 cycles of 95℃ for 30 s, annealing at 50-58℃ for 30 s, and 72℃ for 90 s, then 1 cycle of 95℃ for 1 min, and finally 72℃ for 3 min.PCR products were visualized on a 1% agarose E-Gel 96-well system (Invitrogen), and then purified with 1.0 ul of ExoSAP-IT (GE Healthcare Life Sciences).All products were sequenced in both directions using BigDye Terminator v3.1 (Applied Biosystems) on an ABI 3100 Avant DNA Sequencer (Applied Biosystems) at the Faculty of Science, Kyushu University, Fukuoka.Contigs were made using Vector NTI Advance TM ver.11 (Invitrogen Corp.) and subsequently aligned by eye.Sequence data were assembled and edited in the program Vector NTI Advance Tm ver.11 (Invitrogen Corp.) and MEGA 5 (Tamura et al. 2011).

Phylogenetic inference
The dataset was analyzed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) to explore the strength of the phylogenetic signal under different optimality criteria.
The MP analysis was performed using TNT 1.1 program (Goloboff et al. 2008) with outgroup rooting, default consensus options, Tree Bisection and Reconstruction (TBR) branch swapping, and the default 'traditional search' mode.The tree search employed a parsimony ratchet with 10 000 interactions per run.Parsimony analyses were completed under conditions of equal weighting.Tree analyses were performed with WinClada version 1.00.08 software (Nixon 2002), and consensus cladograms generated from equally parsimonious trees were generated using the same program.Trees were rooted by the outgroup taxon Crematogaster osakensis belonging to the subgenus Orthocrema.
The BI analysis was performed using MrBayes v.3.1.2(Ronquist & Huelsenbeck 2003).For the BI, model selection was limited to those that could be implemented in MrBayes, using the function 'model = mrbayes' in PartitionFinder.The best-fitting partition and substitution models chosen for BI analysis were shown in  (2012c).Divergence times were estimated using the Bayesian method implemented in MCMCTree of PAML4.7 (Yang 2007).Using the approximate likelihood calculation method, the gradient g and Hessian H with BASEML using the GTR + G substitution model were calculated (dos Reis & Yang 2011).The independent rate model for the molecular clock and the GTR + G model for nucleotide substitutions were set in the mcmctree.ctlcontrol file, with the following modification from default setting: substitution rate per time unit = 0.106023; rgene_gamma = 1 10; sigma2_gamma = 1 4.5.

Phylogenetic position and divergence time estimation
The five nuclear gene sequences consisted of 2301 bp and contained 652 variable characters (VC) and 420 parsimony informative characters (PIC) ( As our results from the BI analysis had similar results to the tree inferred by Blaimer (2012c), we use the BI tree to illustrate all our results (Fig. 1).
In MP analysis, the concatenated five-gene dataset yielded 1800 equally parsimonious trees (length = 1407, consistency index = 0.48, retention index = 0.77), of which the strict consensus tree is well resolved.The MP analysis recovered that C. khmerensis sp.nov.was nested within node A with high bootstrap support (100%) (Fig. 2).Further splitting of the node resulted in a trichotomy having branches with C. khmerensis sp.nov., C. borneensis-group and node C.
The ML analysis recovered that C. khmerensis sp.nov.was nested within node A with high bootstrap support (100%) (Fig. 2).Similarly the node displayed a trichotomy among C. khmerensis sp.nov., C. borneensis-group and node C.
The BI recovered that C. khmerensis sp.nov.was nested within node A (Fig. 2).Despite the relatively low level of support (0.88 posterior probability), C. khmerensis sp.nov. at node B was a sister to node C (Fig. 2).

Etymology
The specific name refers to the former kingdom name of Cambodia, the Khmer Empire.

Description
Standing pilosity sparse.Dorsal face of head with appressed setae; one pair of erect setae on frontal lobes.Clypeus with one pair of erect setae.Anterior clypeal margin with two pairs of long setae mixed with short setae laterally.Scape with decumbent setae.Mesontum with appressed setae sparsely.Petiole with one pair of suberect setae posteriorly.Postpetiole with one pair of suberect setae posteriorly.Fourth abdominal tergite with short appressed setae sparsely.

Queen and male
Queens and males of this species are unknown.

Distribution
This species is known only from the type locality in Cambodia (Fig. 5).

Etymology
The specific name is dedicated to Dr. Martin Pfeiffer, who collected the type material.

Description
Petiole elliptical with convex sides, longer than broad; spiracle situated anteriorly midway between dorsal and ventral margin of petiole in lateral view, directed laterally.Subpetiolar process developed acutely.Postpetiole without longitudinal median sulcus, but bilobed behind; spiracle situated anteriorly on lateral surface.Dorsal surface of head mostly smooth or weakly shagreened.Clypeus mostly smooth and shining.Pronotal collar areolately sculptured.Promesonotum weakly punctate.Lateral surface of pronotum smooth and shining.Mesopleuron mostly smooth and shining, but sculptured on surrounding area.Anterodorsal surface of propodeum weakly punctate; posterior half smooth and shining.Lateral surface of propodeum smooth and shining.Dorsal and lateral surfaces of petiole smooth and shining.Dorsal and lateral surfaces of postpetiole smooth and shining.
Standing pilosity sparse.Dorsal face of head with decumbent setae; one pair of erect setae on frontal lobes.Clypeus with one pair of erect setae.Anterior clypeal margin with two to three pairs of long setae mixed with short setae laterally.Scape with appressed setae.Mesonotum with appressed setae sparsely.Petiole with one pair of suberect setae posteriorly.Postpetiole with one pair of suberect setae posteriorly.Fourth abdominal tergite with short appressed setae sparsely.

Queen and male
Queens and males of this species are unknown.

Distribution
This species is known only from the type locality in Malaysia (Borneo) (Fig. 5).

Discussion
Phylogenetic position of C. khmerensis sp.nov.
The MP and ML analyses consistently placed C. khmerensis sp.nov.nested within node A, and forming an unresolved trichotomy with C. borneensis-group and node C (Fig. 2).The reason for this is not clear, but might be due to the large amount of missing data.The BI analysis recovered C. khmerensis sp.nov.nested within node A, and sister to node C, albeit with a low support value.The node A corresponds to node 231 (Blaimer 2012c, fig. 3) of the Australo-Asian Crematogaster clade (Blaimer 2012c, fig. 2, III), the node C corresponds to node 230 (Blaimer 2012c, fig. 3).
In this study, the consensus tree obtained by the BI analysis was used to infer the phylogenetic relationships as our results (Fig. 1) from the BI analysis had similar results to the tree inferred by Blaimer (2012c) Based on molecular and morphological evidence, we treat C. khmerensis sp.nov.as its own species group, the Crematogaster khmerensis-group.In this study, no obvious synapomorphies were identified among C. khmerensis sp.nov.and clade C. Since the existence of undescribed and/or unanalyzed species may decrease the resolution of higher taxonomic levels, the inclusion of more species and morphological characters in both the queen and male castes is considered necessary to corroborate its validity.Further studies should therefore be conducted on closely related taxa to more accurately clarify the position of the C. khmerensis-group.
Although we do not have fresh material of C. pfeifferi sp.nov.for molecular phylogenetic analysis, the species is considered to be most closely related to C. khmerensis sp.nov.and is therefore assigned as a sister species.
Dispersal history of the Australo-Asian Crematogaster clade Blaimer (2012c, fig. 3c) proposed that the Australo-Asian Crematogaster clade evolved in Southeast Asia before colonizing the adjacent Australasian region.Clade C, comprising C. fruhstorferi + C. ss_ AUS5 + C. mjobergi + the C. tetracantha-group, is mostly distributed eastward of Wallace's line, although C. fruhstorferi is also known from Borneo (Blaimer 2012c, CASENT0193728 specimen).The position of C. khmerensis sp.nov., as sister to C. fruhstorferi and the Australasian clade, supports Blaimer's hypothesis of dispersal from the Oriental to the Australian regions (Figs 2, 5), suggesting that sequential dispersal events occurred in different lineages and that they were widespread.
The divergence time estimation and phylogenetic analysis suggest that the most recent common ancestor of C. khmerensis sp.nov.and clade C was inferred to be 15 million years old (Middle Miocene), when the Sundaic region was connected to the extended Indochinese Peninsula (Lohman et al. 2011, fig. 2d).The common ancestor of the C. khmerensis-group was likely distributed over parts of the Sundaic region during periods of low sea level, which would have allowed it to disperse into Indochina and Sundaland before diverging into different taxa as a result of vicariance.The remaining taxa may have dispersed to Sulawesi and Australasian regions east of Wallace's line and diverged.Our divergence time estimations were approximately 5 million years younger than those attained by Blaimer (2012c).While her analysis estimated the node 245 (Blaimer 2012c, fig. 3) at ca 27 Mya, our analysis estimated the same node at ca European Journal of Taxonomy 370: 1-17 (2017) 21 Mya (Fig. 3).This incongruence might be due to the analysis with a subset of her taxon matrix and different method.
Despite extensive field surveys by myrmecologists on the Indochinese Peninsula and Borneo, only two series of the C. khmerensis-group have ever been recorded from Cambodia and Borneo.The members of the C. khmerensis-group are therefore considered to be rare and relatively old taxa with restricted distribution ranges.

Fig. 1 .Fig. 2 .
Fig. 1.Bayesian majority rule consensus tree reconstructed for 90 taxa using five genes (ArgK, CAD, LWRh, Top1, Wg) in a MrBayes analysis.Above node numbers indicate posterior probability.Data were partitioned by PartitionFinder v.1.1.1 and analyzed using a best fit model for each gene and codon position, with 10 million generations and a burn-in of 25 %.Area enclosed by dashed lines is enlarged on Fig. 2.

Fig. 3 .
Fig. 3.Posterior estimates of divergence time of 24 taxa on the phylogenetic tree.Blue bars depict the 95% highest posterior density (HPD).Estimations were performed with MCMCTree using the independent rate model.

Worker
Fig. 4. A-D.Crematogaster khmerensis sp.nov., worker.A. Body in lateral view.B. Full-face view of head.C. Dorsal view of mesosoma.D. Petiole and postpetiole in dorsal view.-E-H.-Crematogaster pfeifferi sp.nov., worker.E. Body in lateral view.F. Full-face view of head.G. Dorsal view of mesosoma.H. Petiole and postpetiole in dorsal view.

Table 2 )
. The MP and ML analyses resulted in similar

Table 1 .
Partitions and models identified by PartitionFinder and used in the maximum likelihood (ML) and Bayesian (BI) analyses.HOSOISHI S. & OGATA K., Phylogenetic position of two new Crematogaster ants topologies for the position of C. khmerensis sp.nov., but the BI analysis differed in topology from the MP and ML analyses.

Table 2 .
Data on number of bases, number of variable characters (VC) and number of parsimony informative characters (PIC).
. The position of C. khmerensis sp.nov. is recovered as a sister to clade C, which HOSOISHI S. & OGATA K., Phylogenetic position of two new Crematogaster ants comprises of C. fruhstorferi + C. ss_AUS5 + C. mjobergi + the C. tetracantha-group, with the basal C. borneensis-group (Fig. 2).While we did not find conclusive evidence of synapomorphies uniting C. khmerensis sp.nov.and clade C in this study, several potential characters are suggested below.Crematogaster fruhstorferi has short, stout propodeal spines and large propodeal spiracles.The propodeal spines of C. fruhstorferi are diverged posteriorly, but not directed vertically as in C. khmerensis sp.nov.The propodeal spiracles of C. fruhstorferi are as large as the base of the propodeal spines, but not 2-3 times as large as in C. khmerensis sp.nov.These morphological data suggest affinity, but are inconclusive.The C. tetracantha-group has laterally-developed pronotal shoulders (Blaimer 2012b), especially C. dahlii and C. tetracantha, which have pronotal shoulders distinctly developed as spines.The presence of laterally-developed pronotal shoulders suggests a close affiliation between C. khmerensis sp.nov.and the members of the C. tetracantha-group, even though C. fruhstorferi, C. mjobergi and C. ssAUS5 do not have developed pronotal shoulders.The phylogenetic relationship inferred by molecular analysis indicates that laterally-developed pronotal shoulders evolved independently and in two steps, i.e., once in C. khmerensis sp.nov.and also at base of the C. tetracantha-group, or it is also parsimonious that the shoulders were gained in the ancestor of the clade B then lost in the C. mjobergi and C. ssAUS5 clade.