Comprehensive Description

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Anochetus boltoni Fisher HNS ZBK sp. nov. 534E91EF7FBB Figures: worker 2a,b, 5a; male 2c,d, 8a; map 6a

Type Material: Holotype worker, MADAGASCAR : Antsiranana , Parc National de Marojejy , Manantenina River, 28.0 km 38 ° NE Andapa , 8.2 km 333° NNW Manantenina , 14°26'12"S , 049°46'30"E , 450 m , sifted litter, rainforest, 12-15 Nov 2003 (coll. B. L. Fisher et al.), comma collection code: BLF08985 pin code: CASENT0104542 ( CASC ) . Paratype . 8 workers with same data as holotype but pins coded, CASENT0487895 , CASENT0487896 , CASENT0487897 , CASENT0006943 . ( BMNH , MCZ , CAS ) ; CO1 Barcode from paratype collection and coded CASENT0487895-D01

Figure 2. Anochetus HNS spp. full face and lateral view. A-B, boltoni HNS worker CASENT0104542. C-D, boltoni HNS male CASENT0063847. E-F, goodmani HNS worker CASENT0104543. G-H, goodmani HNS ergatoid queen CASENT0454531. doi:10.1371/journal.pone.0001787.g002

Figure 3. Anochetus grandidieri HNS full face and lateral view. A-B, large worker CASENT0497580. C-D, small worker CASENT0033463. E-F, large queen CASENT0041177. G-H, small queen CASENT0498467. I -J, male CASENT0049858. doi:10.1371/journal.pone.0001787.g003

Worker measurements: maximum and minimum based on all specimens, n= 20, (holotype): HL 1.80-2.08 (1.95), HW 1.61- 1.89 (1.71), CI 87-98 (88), EL 0.33-0.41 (0.36), ML 1.15-1.25 (1.20), MI 59-66 (61), SL 1.83-1.96 (1.84) SI 101-115 (107), WL 2.63-2.89 (2.73), FL 1.97-2.13 (2.03), PW 0.95-1.06 (1.00).

Male measurements: maximum and minimum based on n = 2 from Madagascar: HL 0.89-0.91, HW 1.05-1.13, CI 118- 125, EL 0.56-0.62, SL 0.24, SI 21-23, WL 2.20-2.24, FL 1.75-1.80

Worker Diagnosis: Blade of mandible with five teeth and denticles located along distal two thirds of blade's length. Propodeum with short teeth (Fig. 5a). Dorsolateral margin of petiole with long spine (Fig. 5a). In frontal view, petiolar margin deeply U-shaped. Pilosity, sculpture as in Figures 2a,b.

Male caste: Dorsolateral margin of petiole with acute spine. The species is most similar to A. goodmani HNS , but can be easily distinguished by its petiole node with a pair of large apical spines.

Distribution and biology. The distribution is limited to collections made between 450 m and 750 m in rainforest in Parc National de Marojejy and 240 m from Ambanitaza near Antalaha (Fig. 4a). It has been collected three times in rotten logs and once in a leaf litter sample. Males have been collected in malaise samples on 20-25 Dec 2004 at 488 m in Parc National de Marojejy CO1. The two populations where collections have been made to date are characterized by a deep divergence within the DNA barcode region (Maximum - 8%) (Fig. 15).

Diagnostic barcoding loci. A. boltoni HNS : ATCT-42-45 & RTTAR-66-70

Discussion: Specimens from Ambanitaza differ notably in shape of propodeal spines and length of spines on petiole from those of the type locality. Though these localities are quite close (40 km apart), these character differences are noticeable, and correspond to significant differences in CO1 (34 base pairs) and ITS1. While specimens from each location were invariant within 18S , there is a 7 bp insertion within ITS1 that is characteristic of the Ambanitaza population which is missing from all specimens from Marojejy. Ultimately, more collections need to be made and evaluated in order to test the hypothesis that these populations represent separate species. One important factor to consider in the testing of that hypothesis is reproductive strategy, which is, to our knowledge , through fission. Though the queen caste is not known, based on overall similarity of workers with A. goodmani HNS , it is likely that the queen of boltoni HNS is wingless. Queens have never been collected during the 12 month malaise trap sampling even though males were collected. Species that reproduce by fission may show greater geographic differences in morphology and CO1.

Figure 4. Anochetus madagascarensis HNS full face and lateral view. A-B, worker CASENT0104547. C-D, queen CASENT0498419. E-F, male CASENT0049282. doi:10.1371/journal.pone.0001787.g004

Figure 5. Anochetus HNS workers, upper part of petiole from front view. A, boltoni HNS CASENT0104542. B, goodmani HNS CASENT0104543. C, grandidieri HNS (large form) CASENT0497580. D, madagascarensis HNS CASENT0498309. doi:10.1371/journal.pone.0001787.g005

Additional material examined for Anochetus boltoni HNS : In addition to the type material, specimens from 4 additional collecting events from the following three localities were examined in this study. MADAGASCAR : Province Antsiranana : Parc National de Marojejy , Manantenina River, 27.6 km 35° NE Andapa ; Parc National de Marojejy , Manantenina River, 28.0 km 38° NE Andapa ; Foret Ambanitaza , 26.1 km 347° Antalaha . This material shows greater variation in number of denticles along blade of mandible, ranging from 5-7, compared to the paratypic material.


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