Description and DNA barcoding of Crematogaster fraxatrix Forel, 1911 and two new closely related species from Cambodia and Indonesia (Hymenoptera, Formicidae)

Abstract Crematogaster fraxatrix Forel, 1911 and two new species, C. chhangi sp. n. and C. simboloni sp. n., are described from Cambodia and Indonesia, respectively. DNA sequences were generated for C. fraxarix and the two newly described species using 3 amplications of two regions of the mitochondrial gene COI with a total of 1129 bp. The mean interspecific divergences are 9.4% and 23.5% for C. fraxatrix vs. C. chhangi, C. simboloni, respectively. DNA sequences reveal that C. simboloni is found to be genetically distinct from the other two species, but C. chhangi is not distinct from C. fraxatrix.


Introduction
Crematogaster fraxatrix was described by Forel (1911) based on the worker specimens from Malaysia (Borneo). This species is presently assigned to the subgenus Crematogaster (Blaimer 2012b). A recent molecular work re-classified the former sixteen subgenera into two larger subgenera, Crematogaster and Orthocrema (Blaimer 2012b). The subgenus Crematogaster is the largest, including more than 220 species, its workers generally have anteriorly broader petiole ('flared'-shape in Blaimer 2012b), but C. ferrarii, C. fraxatrix and C. ransonneti uniquely have the petiole broader in the middle portion among Asian Crematogaster fauna. The close relationship between C. ferrarii and C. fraxatrix was also suggested by a molecular phylogeny (Blaimer 2012c). However, C. fraxatrix can be easily distinguished by the densely sculptured mesopleuron from C. ferrarii, by the weakly concave metanotal groove from C. ransonneti, respectively. In the course of our recent examination of Crematogaster specimens collected from southeast Asia, two distinct species related to C. fraxatrix were found, which are here described as new species. Cytochrome oxidase I (COI) sequence data from Crematogaster fraxatrix was further compared with that of the two new species. DNA barcodes have been recently used in biodiversity studies of ant species (Smith et al. 2005), and are used as an additional and powerful method in integrative taxonomy (Schlick-Stiner et al. 2010). They can thus provide a useful reference for the identification of Asian Crematogaster species. Our analysis included not only in the conventional 5' DNA barcoding region, but also the 3' region of COI region. The relationship between C. fraxatrix and the two new species is discussed, based on morphological features and sequence divergence.

Sources of material and abbreviations
Specimens were examined and/or deposited in the collections listed below. Codes for public institutions mainly follow those in Brandão (2000). Nest series samples, most of which were recently collected, are represented as colony codes, e.g., "SH12-Cam-70."

Measurements and indices
Most observations were made using an Olympus SZX12 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 were made with an Olympus SZX12 stereomicroscope using micrometers. All measurements are expressed in millimeters, recorded to the second decimal place. The measurements for petiole and postpetiole follow Longino (2003).

Genetic analysis
Genomic DNA was extracted from tissues rich in mitochondria (e.g. legs) using a DNeasy Blood & Tissue kit (Qiagen, Maryland, USA). A 497 bp region of the mitochondrial genome, including barcoding regions of the cytochrome oxidase I (COI) was amplified via the polymerase chain reaction (PCR) using the following combinations of the primers, "LepF1" 5'-ATTCAACCAATCATAAAGATATTGG-3' and "C_ANTMR1D-RonIIdeg_R" 5'-GGRGGRTARAYAGTTCATCCWGTWCC-3' (used only for PCR), and "MLepF1" 5'-GCTTTCCCACGAATAAATAATA-3' and "LepR1" 5'-TAAACTTCTGGATGTCCAAAAAATCA-3' (Hebert et al. 2004;Fisher and Smith 2008;Hajibabaei et al. 2006). Reactions were carried out at 10 µl volumes in a PCR Thermal Cycler MP (TaKaRa Bio Inc.) under the following conditions: a first cycle of 94°C for 2 min, followed by 5 cycles of 94°C for 40 sec, annealing at 45°C for 40 sec, and 72°C for 1min, then 36 cycles of 94°C for 40 sec, annealing at 51°C for 40 sec, and finally 72°C for 1 min for the COI. A 632 bp region of the 3' region of COI was amplified via the polymerase chain reaction (PCR) using primers "Jerry" 5'-CAACATTTATTTTGATTTTTTGG-3' and "Pat" 5'-TCCAATGCACTAATCT-GCCATATTA-3' (Simon et al. 1994). Reactions were carried out at 10 µl 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, 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 µl of ExoSAP-IT (GE Healthcare Life Sciences). All products were sequenced in both directions (except for C_ANTMR1D-RonIIdeg_R) using BigDye Terminator v3.1 (Applied Biosystems) on an ABI 3100 Avant DNA Sequencer (Applied Biosystems) at the Faculty of Science, Kyushu University, Fukuoka. Some fragments were removed prior to alignment, due to low quality. After trimming, the 5' DNA barcoding region sequenced in this study were 497bp, therefore these sequences were not strictly DNA barcodes. Using the three primer sets, non-overlapping fragments of 244, 253 and 632 bp were sequenced respectively. DNA sequence data for eight individuals of three Crematogaster species were thus generated, and deposited at DNA Data Base of Japan, DDBJ (with accession numbers shown in Table 1). All data were registered in the project called: "Crematogaster ants in Asia" (CREAA) on Barcode of Life Database (BOLD). Among the three species, C. chhangi was sequenced from one nest series (SH12-Cam-70) from Cambodia, and C. simboloni was successfully sequenced only from one Krakatau specimen. 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 (Kimura 1980) distances with MEGA 5 (Tamura et al. 2011). The phylogenetic tree was estimated using Neighbor-Joining (NJ) (Saitou and Nei 1987) in the program MEGA 5.
The neighbor-joining tree ( Fig. 9) shows Crematogaster chhangi sister to C. fraxatrix, with high bootstrap support (100%). Among the three species examined, C. simboloni was distinctly separated from the C. chhangi and C. simboloni with higher genetic divergence: 24.3 % to C. chhangi and 22.6 % to 24.6 % to C. fraxatrix. Crematogaster chhangi is distinguished from C. fraxatrix only in having an acutely developed subpetiolar process, whereas C. simboloni is quite different from C. chhangi and C. fraxatrix in having a densely sculptured promesonotum. Diagnosis. This species is similar to C. fraxatrix, but can be distinguished by the dorso-ventrally flattened propodeal spiracles and acutely developed subpetiolar process in the worker caste. The COI divergence between C. chhangi and C. fraxatrix did not seem relatively high (8.1-10.8 % K2P distances) (cf. Blaimer 2012a), but the two species are clearly separated from each other by the characters shown above.

Key to species of Crematogaster fraxatrix-group
Worker description. Workers presumably monomorphic. Posterior corners of head rounded. Anterior clypeal margin slightly concave in the median portion. Compound eyes not projecting beyond lateral margins of head in full face view. Scape reaching posterior corner of head. Antennal club 3-segmented. Pronotal dorsum with distinct ridges laterally. Mesonotal dorsum with lateral ridges. Mesonotum not higher than pronotum in lateral view; forming same dorsal outline with pronotum in lateral view. Metanotal groove straight in dorsal view, deep and forming a concave region between mesonotum and propodeum. Propodeal spiracles oval, flattened dorso-ventrally, located on the lateral sides of propodeum; the horizontal diameter more than two times larger than the vertical diameter. Propodeal spines developed long, directed upward and straight. Petiole broader in the middle portion. Subpetiolar process acutely developed. Postpetiole weakly bilobed, but without longitudinal median sulcus. Petiole slightly wider than postpetiole in dorsal view. Erect pilosity sparse. Scape with abundant erect to suberect setae. Dorsal face of head with suberect setae. Clypeus with suberect setae; one pair of longer setae directed medially on anteriormost portion. Anterior clypeal margin with one single setae and one pair of longer setae, mixed with some shorter setae on the sides. Mesosoma with sparse erect setae. Fourth abdominal tergite with erect to suberect sparse setae. Dorsal surface of head generally smooth and shining, but feeble rugulae between frontal carinae; longitudinal rugulae surrounding antennal sockets and on gena. Clypeus weakly striated with longitudinal rugulae. Promesonotum striated with feeble rugulae. Lateral surface of pronotum smooth and shining. Mesopleuron sculptured. Lateral surface of propodeum generally smooth, but with feeble rugulae on the lower portion. Body color brown.
Distribution. This species is known only from the type locality of Cambodia.
Etymology. This species is dedicated to Mr. Phourin Chhang, Forestry Administration of Cambodia, who helped with field surveys in Cambodia. Diagnosis. This species is similar to C. chhangi, but can be distinguished by the oval-shaped propodeal spiracles and weakly developed subpetiolar process in the worker caste. Based on COI divergence, the specimens from Peninsular Malaysia were separated from the Bornean specimens with a high support value (Fig. 9). This is presumably due to lack of gene flow between the populations, but they showed no distinct morphological differences between each other. The COI divergence of 0-9.3 % (K2P distances) was recorded within C. ranavalonae clade in Madagascar (Blaimer 2012a). Further geographic sampling is therefore needed to determine whether the variation of 0.4-6.9 % (K2P distances) represents the intraspecific variation or includes some interspecific variation.
Worker description. Workers with weak polymorphism in size. Posterior corners of head rounded in smaller worker, but squared in larger workers. Anterior clypeal margin slightly concave in the median portion. Compound eyes not projecting beyond lateral margins of head in full face view. Scape reaching posterior corner of head. Antennal club 3-segmented. Pronotal dorsum with distinct ridges laterally. Mesonotal dorsum with lateral ridges. Mesonotum slightly higher than pronotum in larger workers in lateral view. Metanotal groove straight in dorsal view, deep and forming a concave region between mesonotum and propodeum. Propodeal spiracles dorso-ventrally oval, located on lateral sides of propodeum; the horizontal diameter slightly larger than the vertical diameter even in smaller workers. Propodeal spines long, directed upward and straight. Petiole broader in the middle portion. Subpetiolar process developed as small, blunt denticle. Postpetiole weakly bilobed, but without longitudinal median sulcus. Petiole slightly wider than postpetiole in dorsal view.
Sparsely hirsute with erect setae. Scape with abundant erect to suberect setae. Dorsal face of head with suberect setae. Clypeus with suberect setae; one pair of longer setae directed medially on anteriormost portion. Anterior clypeal margin with one single setae and one pair of longer setae, mixed with some shorter setae on the sides. Mesosoma with sparse erect setae. Fourth abdominal tergite with sparse erect to suberect setae.
Dorsal surface of head generally smooth and shining, but feeble rugulae between frontal carinae; longitudinal rugulae surrounding antennal sockets and on gena. Clypeus weakly striated with longitudinal rugulae. Pronotum striated with feeble rugulae. Mesonum weakly striated with feeble rugulae. Lateral surface of pronotum smooth and shining. Mesopleuron sculptured, but the central portion relatively smooth. Lateral surface of propodeum with feeble rugulae.
Body color reddish-brown to black. Distribution. This species is known from southern Thailand and Malaysia (Peninsular and Borneo).  Diagnosis. This species is similar to C. chhangi and C. fraxatrix, but can be easily distinguished from these by the sculptured promesonotum in the worker caste. The COI divergence between C. simboloni and C. chhangi (24.3 % K2P distances), as well as C. simboloni and C. fraxatrix (22.6 to 24.6 % K2P distances) were also high.
Worker description. Workers monomorphic. Posterior corners of head rounded. Anterior clypeal margin slightly concave in the median portion. Compound eyes projecting slightly beyond lateral margins of head in full face view. Scape reaching posterior corner of head. Antennal club 3-segmented. Pronotal dorsum with distinct ridges laterally. Mesonotal dorsum with lateral ridges. Mesonotum slightly higher than pronotum in lateral view. Metanotal groove straight in dorsal view, deep and forming a concave region between mesonotum and propodeum. Propodeal spines long, directed upward and straight. Propodeal spiracles oval, flattened dorso-ventrally, located on the lateral sides of propodeum, or the postero-lateral corners; the horizontal diameter slightly larger than the vertical diameter. Petiole broader in the middle portion. Subpetiolar process undeveloped. Postpetiole weakly bilobed with feeble median sulcus. Petiole as wide as postpetiole in dorsal view. Sparsely hirsute with erect setae. Scape with abundant erect to suberect setae. Dorsal face of head with suberect setae. Clypeus with suberect setae; one pair of longer setae medially on anteriormost portion. Anterior clypeal margin with one single setae and one pair of longer setae, mixed with some shorter ones on the side. Mesosoma with short and sparse erect setae. Fourth abdominal tergite with few erect to suberect setae. Dorsal surface of head generally smooth and shining, but feeble rugulae between frontal carinae; longitudinal rugulae surrounding antennal sockets and on gena. Clypeus striated with longitudinal rugulae. Pronotum striated with longitudinal rugulae with the sculptured space; the longitudinal rugulae separated from anterior mesonotal margin. Mesonotum sculptured. Lateral surface of pronotum smooth and shining. Mesopleuron sculptured, but the central portion relatively smooth. Propodeal dorsum sculptured anteriorly. Lateral surface of propodeum weakly sculptured and striated with feeble rugulae. Body color brown.
Distribution. This species is known only from Indonesia (Krakatau).
Etymology. This species is dedicated to Dr. Herwint Simbolon, Research Centre for Biology, Lembaga Ilmu Pengetahuan Indonesia (The Indonesian Institute of Sciences), who collected the type material.