Taxonomy and DNA Sequencing of Crematogaster coriaria Mayr , 1872 ( Hymenoptera : Formicidae ) , with Redescriptions of the Worker , Queen , and Male Castes

Crematogaster coriaria Mayr, 1872, is revised based on the nest series samples. The following new synonymies are proposed: C. coriaria Mayr, 1872 = C. coriaria fraudatrix Forel, 1911 syn. nov. = C. spengeli Forel, 1912 syn. nov. = C. spengeli taipingensis Forel, 1913 syn. nov. Worker, queen, and male castes of C. coriaria Mayr, 1872, are redescribed. The polymorphism of C. coriaria Mayr, 1872, was also discussed. DNA sequences were generated for C. coriaria Mayr, 1872, using an amplification of second half region of the mitochondrial gene COI with a total of 711 bp. The interspecific COI divergence is 17.4% for C. coriaria Mayr, 1872, and C. modiglianii Emery, 1900.


Introduction
Crematogaster coriaria Mayr, 1872, was described by Mayr [1] based on the worker specimens from Borneo, Malaysia.One following subspecies of C. coriaria Mayr, 1872, has been recognized: C. coriaria fraudatrix Forel, 1911, by Forel [2] based on the workers from Sumatra, Indonesia.This species had been classified into the former subgenus Paracrema but presently assigned to the subgenus Crematogaster [3].
Crematogaster coriaria Mayr, 1872, is distributed in Malaysia (Peninsula, Borneo) and Indonesia (Sumatra, Java) and has been reported from several localities in the literatures [4][5][6].This species inhabits well-developed forests and nests in the dead woods.
This species is relatively roughly distinguished from other Crematogaster fauna by 4-segmented antennal club and weak sculptured body surface.However, due to the strong polymorphism, the taxonomy of this species has been confused as follows.This species is distinctly polymorphic in size and sculpture, causing some synonymies in some species and subspecies.Crematogaster coriaria Mayr, 1872, has relatively smooth surface of body in smaller workers but develops the rugulae and sculpture in larger workers.
The present paper aims to revise the synonymies based on the nest series samples.Such samples can reveal morphological continuities within conspecific taxa.
As in other ant genera, taxonomy of the genus Crematogaster has been generally improved based on the worker caste [7].The sexual forms are poorly investigated because of the rarity of sexual castes in the collections and unexplored characters in the genus (but see [8,9]).The present paper describes queen and male castes of C. coriaria Mayr, 1872, and it will provide a starting point for taxonomic studies to find the valid characters.
Molecular approaches including DNA barcodes have been recently used in biodiversity studies of ant species [10] and are used as an additional and powerful method in integrative taxonomy [11].They can thus provide a useful reference for the identification of Asian Crematogaster species.Therefore, cytochrome oxidase I (COI) sequence data from Crematogaster coriaria Mayr, 1872, is also generated and compared with the closely related species, C. modiglianii Emery, 1900.Our analysis included the 3  region of COI region.
In the present paper, based on our examination of type materials of almost all of the forms under consideration Psyche and nontype specimens including nest series samples, we revise their names and solve synonymy of taxa related to the species.In addition, this study redescribes the queen and male castes of the species and then contributes toward the taxonomy on sexual forms of Crematogaster ants in specieslevel identification.The sequence data provide additional information for the species.

Materials and Methods
2.1.Sources of Material.Type and nontype specimens were examined and/or deposited in the collections listed below.Codes for public institutions generally follow those in [12].KUEC: Institute of Tropical Agriculture, Kyushu University, Fukuoka, Japan.MHNG: Musee d'Histoire Naturelle, Geneva, Switzerland.

Observation.
Most observations were made at various magnifications on a Leica M205C stereomicroscope.Images were taken using a Canon EOS 50D with Canon MP-E 65 mm 1-5x Macro Lens and then processed using Combine ZM.

Measurements and Indices.
Measurements were made under a Leica M205C stereomicroscope using micrometers.All measurements are expressed in millimeters, recorded to the second decimal place.The measurements for petiole and postpetiole follow [13] and those for queen and male specific measurements follow [8].
Data were plotted on log-log scales and regression lines calculated for workers using R version 2.14.2.
Head width (HW): maximum width of head in fullface view, excluding the eyes, in the worker and queen castes, but measured including the eyes in the male caste.Head Length (HL): perpendicular distance from vertex margin to line tangent anteriormost projections of clypeus in full-face view.
Scape length (SL): length of the first antennal segment, excluding the neck and basal condyle.
Eye Length (EL): maximum length of the compound eye.
Pronotal Width (PW): maximum width of the pronotum in dorsal view in the worker caste.
Weber's length of the mesosoma (WL): diagonal length, measured in lateral view from the anterior margin of the pronotum (excluding the collar) to the posterior extremity of the propodeal lobe.
Propodeal Spine Length (PSL): measured from tip of propodeal spine to closest point on outer rim of propodeal spiracle in the worker caste but measured from tip of propodeal spine to propodeal declivity in lateral view in the queen caste.
Petiole Length (PtL): length of the petiole in lateral view.
Petiole Width (PtW): maximum width of petiole in dorsal view.
Petiole Height (PtH): height of the petiole in lateral view.
Queen and male specific measurements [8] are as follows: Mesonotal width (MSNW): maximum width of mesonotum in dorsal view.

Genetic Analysis.
Genomic DNA was extracted from tissues rich in mitochondria (e.g., legs) using a DNeasy Blood & Tissue kit (Qiagen, Maryland, USA).A 711 bp region of the mitochondrial genome, 3  region of the cytochrome oxidase I (COI), was amplified via the polymerase chain reaction (PCR) using primers "Jerry" 5  -CAA-CATTTATTTTGATTTTTTGG-3  and "Pat" 5  -TCCAAT-GCACTAATCTGCCATATTA-3  [14].Reactions were carried out at 10 L volumes in a PCR Thermal Cycler MP (TaKaRa Bio Inc.) under the following conditions: a first cycle of 94 ∘ C for 1 min, followed by 5 cycles of 94 ∘ C for 1 min, annealing at 48 ∘ C for 90 s, and 72 ∘ C for 90 s, and then 30 cycles of 94 ∘ C for 1 min, annealing at 51 ∘ C for 90 s, and finally 72 ∘ C for 90 s for the COI.
PCR products were visualized on a 1% agarose E-Gel 96-well system (Invitrogen) and then purified with 1.0 L of ExoSAP-IT (GE Healthcare Life Sciences).All products were sequenced in both directions using BigDye Terminator v3.one nest series (SH10-Mal-18) from Ulu Gombak, Malaysia (Peninsular Malaysia).Contigs were assembled using Vector NTI Advance TM ver.11 (Invitrogen Corp.) and subsequently aligned by eye.Genetic distances were estimated using the Kimura-2-parameter [15] distances with MEGA 5 [16].
Petiole scoop shaped, but flattened with convex sides or broader anteriorly, longer than broad; spiracle situated anteriorly midportion between dorsal and ventral margin of petiole in lateral view, directed laterally.Postpetiole without distinctly longitudinal median sulcus, but bilobed behind in large workers; spiracle situated anteriorly on lateral surface.
Posterior half of head sculptured without rugulae; gena with rugulae on surrounding antennal sockets.Clypeus with abundant longitudinal rugulae in large workers (Figures 1(b) and 2(c)), but weakly punctuated in small workers (Figure 2(b)).Promesonotum weakly punctuated.Lateral surface of pronotum smooth and shining.Mesopleuron densely sculptured.Anterodorsal surface of propodeum generally sculptured.Lateral surface of propodeum smooth and shining.Dorsal surface of petiole smooth and shining.Lateral surface of petiole weakly punctuated.Dorsal surface of postpetiole sculptured.Lateral surface of postpetiole generally smooth and shining.
Standing pilosity sparse.Dorsal face of head with some pairs of long and erect setae.Clypeus with some pairs of erect setae.Anterior clypeal margin with two pairs of long setae mixed with short setae laterally.Mesonotal dorsum with sparse erect setae.Petiole with one pair of suberect setae posteriorly.Postpetiole with one pair of suberect setae posteriorly.Fourth abdominal tergite with appressed setae and sparse erect setae.
Body color yellow-brown.
Queen Measurements and Indices.HW 1.54-1.59;HL Masticatory margin of mandibles with five teeth.Antennal scapes reaching posterior margin of head.
Dorsal surface of head with longitudinal rugulae, but posterior half weakly striated (Figure 3(b)).Mandibles generally smooth.Clypeus with abundant longitudinal rugulae.Mesonotum weakly shining, but weakly sculptured (Figure 3(c)).Lateral surface of pronotum and mesopleuron with feeble longitudinal rugulae (Figure 3(d)).Dorsal surface of propodeum with longitudinal rugulae (Figure 3(c)).Lateral surface of propodeum with distinct longitudinal rugulae (Figure 3(d)).Dorsal surface of petiole weakly sculptured (Figure 3(c)).Lateral surface of petiole sculptured (Figure 3(d)).Dorsal and lateral surface of postpetiole sculptured (Figure 3(c  Compound eyes large and protruding, midline of eyes situated well below midline of head.Antennae 12-segmented; flagellar segment longer than broad in apical to second basal segments (antennal segments III to XII).Head much wider than long (CI 137).In full-face view ocellar triangle situated at posterior head margin and elevated with respect to rest of face.Occipital carinae distinct.
Mesonotum in dorsal view longer than broad (MSNI 61.2); scutellum in dorsal view broadly tapering from anterior to posterior end, dorsoposteriorly rounded.Metanotum not protruding from below scutellum.Dorsal face of propodeum almost as long as posterior face.Propodeal spines absent.Petiole in dorsal view oval, weakly convex laterally, in lateral view anteriorly tapering.Subpetiolar process absent.Postpetiole almost round, but weakly bilobed posteriorly, lacking median impression.
Color brown.
Distribution and Biology.This species is distributed in Malaysia (Peninsula, Borneo) and Indonesia (Sumatra, Java) (Figure 5).This species inhabits well-developed forests and nests in the dead woods.
1 (Applied Biosystems) on an ABI 3100 Avant DNA Sequencer (Applied Biosystems) at the Faculty of Science, Kyushu University, Fukuoka.It is noted that these sequences are not DNA barcoding region.DNA sequence data for one individual of Crematogaster coriaria Mayr, 1872, and closely related species, C. modiglianii Emery, 1900, were thus generated and deposited at DNA Data Base of Japan, DDBJ (with accession numbers: LC068833, for C. coriaria Mayr, 1872, and LC068834 for C. modiglianii Emery, 1900).
Crematogaster coriaria Mayr, 1872, was sequenced from one nest series (SH10-Mal-85) from Ulu Gombak, Malaysia (Peninsular Malaysia), and C. modiglianiiEmery, 1900, from )). Dorsal surface of head with sparse erect setae and abundant appressed setae.Anterior clypeal margin with one to two pairs of long setae mixed with short setae laterally.Scape with decumbent to appressed long setae.Mesonotal dorsum with erect setae sparsely.Fourth abdominal tergite with erect setae and appressed setae sparsely.Description of Male.Mandibles very short, masticatory margin with two teeth, one large apical, but a blunt second lobelike.Clypeus weakly convex with almost straight median.