Taxonomy and species-groups of the subgenus Crematogaster (Orthocrema)... 23 Taxonomy and species-groups of the subgenus Crematogaster (Orthocrema) in the Malagasy region (Hymenoptera, Formicidae) Bonnie B. Blaimer1,† 1 Department of Entomology, University of California-Davis, One Shields Ave, Davis, CA 95616, USA † urn:lsid:zoobank.org:author:84EADAC0-B6E3-4B0A-9A66-1C4DDD6E5C1B Corresponding author: Bonnie B. Blaimer (bbblaimer@ucdavis.edu, bonnieblaimer@gmail.com) Academic editor: Brian Fisher  |  Received 7 January 2012  |  Accepted 29 May 2012  |  Published 4 June 2012 urn:lsid:zoobank.org:pub:E032502C-AC0D-427B-AAE9-451294BD2911 Citation: Blaimer BB (2012) Taxonomy and species-groups of the subgenus Crematogaster (Orthocrema) in the Malagasy region (Hymenoptera, Formicidae). ZooKeys 199: 23–70. doi: 10.3897/zookeys.199.2631 Abstract The species-level taxonomy of the subgenus Crematogaster (Orthocrema) in the Malagasy region is evalu- ated with both morphological data from worker and queen ants, and genetic data from three nuclear markers (long wavelength rhodopsin, arginine kinase and carbamoylphosphate synthase). These two types of data support the existence of six Orthocrema species: Crematogaster madecassa Emery, C. rasoherinae Forel, C. telolafy sp. n., C. razana sp. n., C. volamena sp. n. and C. mpanjono sp. n.. Two new synonyms of C. rasoherinae Forel are recognized, C. rasoherinae brunneola Emery, syn. n. and C. voeltzkowi Forel, syn. n., as these were not supported as distinct taxa by the data. A neotype is designated for C. rasoherinae lec- totypes are designated for C. madecassa, C. rasoherinae brunneola and C. voeltzkowi. Species descriptions, images, distribution maps and identification keys based on worker and queen ants are given for all six species. A diagnosis of the subgenus Orthocrema in the Malagasy region is presented for both workers and queens. Within the Malagasy Orthocrema, three distinct phylogenetic lineages are suggested by molecular and morphological data. Newly defined monophyletic species-groups are thus the C. madecassa-group (C. madecassa, C. telolafy and C. razana) and the C. volamena-group (C. volamena and C. mpanjono) C. raso- herinae represents an isolated lineage in the Malagasy region and its closest relatives remain unclear. Other interesting biological findings are the presence of an intermediate caste between workers and queens in C. rasoherinae and C. madecassa, and unusually large workers in C. volamena resembling a major caste. Keywords Crematogaster, Orthocrema, Madagascar, Indian Ocean islands, taxonomy, intermediate workers ZooKeys 199: 23–70 (2012) doi: 10.3897/zookeys.199.2631 www.zookeys.org Copyright Bonnie B. Blaimer. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC- BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ReseARCH ARTiCle Launched to accelerate biodiversity research A peer-reviewed open-access journal

Bonnie B. Blaimer / ZooKeys 199: 23–70 (2012) 24 introduction The large and species-rich ant genus Crematogaster Lund ( 450 described species Bol- ton 2011) has a global distribution throughout most forest and savannah habitats in warm-temperate to subtropical and tropical climates. Crematogaster species diversity is highest in the tropics and subtropics however, where these ants often form a dominant part of the local ant community. The majority of Crematogaster species nest arboreally, for example in twigs or under bark, but ground nesting seems to be equally common in temperate and cooler climates and also more prevalent in some species-groups in the tropics. The species-level taxonomy of Crematogaster ants is notoriously difficult and encumbered by synonyms and ambiguous subspecies names (Longino 2003 Ward 2010). Blaimer (2010) provided a comprehensive review on the natural history and taxonomic state of Crematogaster. In the Malagasy region, here defined as Madagascar and the surrounding Indian Ocean islands, the taxonomy of Crematogaster is currently being revised in a series of publications, subdivided into the several distinct species-groups present in the region (see Blaimer 2010, 2012). The total species diversity of Crematogaster in the Malagasy region is estimated to be approximately 33 species (Blaimer, unpublished data). This estimate represents a mixture of previously described species, species new to science, and reductions due to synonymy. Most of these species occur only in Madagascar, but five species also are found on the Comoros Islands, Mayotte and the Seychelles. Recent intensive inventories of arthropods and especially the ant fauna in the Malagasy region (see e.g. Fisher and Penny 2008) have immensely increased the extent of available specimens for revisionary work, generating much more complete distribution records for already described species and discovering numerous undescribed new species. The present study is part of this larger revisionary work and treats all species associ- ated with the subgenus Orthocrema Santschi (1918) in the Malagasy region. Recent molecular work (Blaimer, in prep.) has found Malagasy species placed in the subgenus Mesocrema Santschi (1928) to be closely related to the former, and these are therefore included with Orthocrema in the present revision. This altered classification follows anticipated changes in the subgeneric classification of Crematogaster in the near future, based upon a molecular phylogenetic framework (Blaimer in prep.). Up to now, one species has been described from the Malagasy region for Or- thocrema, C. madecassa Emery, whereas three species and subspecies have been de- scribed for Mesocrema: C. rasoherinae Forel, C. rasoherinae brunneola Emery and C. voeltzkowi Forel. The latter has been recorded exclusively from the Comoros Islands, whereas the other species were first described from Madagascar. My observations suggest that the Malagasy Orthocrema present an exception to the predominantly arboreal life habit of Crematogaster ants in this region. Most species in this group appear to be generalists, as they have been collected nesting both on the ground in rotten logs or branches, or arboreally in dead twigs or bark and canopy moss mats. A very interesting aspect of the biology of some of the Malagasy species in this group is the presence of intermediate workers in the colony. These possess morphological

Taxonomy and species-groups of the subgenus Crematogaster (Orthocrema)... 25 features that are intermediate between workers and queens, but their function and behavior in the colony remains unclear. Intermediates have also been reported in the North American Crematogaster (Orthocrema) species C. smithi, where they were denoted as ‘large workers’ (Heinze et al. 1999). In the case of the latter, it was shown that these had the ability to lay unfertilized trophic eggs, but were not capable of sexual reproduction (Heinze et al. 1999, 2000). The presence of this separate caste may be a more widespread phenomenon in Orthocrema species. In the following, I focus on a reevaluation of the presently described Malagasy Orthocrema species with both morphological and molecular methods, and further de- scribe new species that are supported by these two types of data. A second aim of this study is the delimitation of two morphologically and genetically distinct species- groups within Malagasy Orthocrema. Materials Morphological study All morphological observations were made with a Leica MZ12.5 stereomicroscope. Standard measurements (in mm) were taken at 50× with a Wild M5A stereomicro- scope and a dual-axis Nikon micrometer wired to a digital readout. Measurements are given to the second decimal place, and indices are presented as decimal fractions (also to the second decimal). Ranges are always expressed as minimum – maximum values. Measured specimens were chosen to represent the entire distribution range of a given species. The abbreviations used for measurements and indices below follow Blaimer (2010) and Longino (2003) for illustrations of these see Blaimer (2010). Measurements and indices HW Maximum head width including eyes, in full face view. HL Head length perpendicular distance from line tangent to rearmost points of vertex margin to line tangent to anterior most projections of clypeus, in full face view. EL Eye length measured along the maximum diameter. SL Scape length length of scape shaft from apex to basal flange, not includ- ing basal condyle and neck. If scape is strongly arched, this measurement is taken as the chord length from the basal flange to the apex. PTL Petiole length measured in lateral profile as the distance from dorsopos- terior margin of segment to anterior inflection point where petiole curves up to condyle. PTH Petiole height measured in lateral profile as vertical distance from ventral margin to highest point of dorsoposterior margin.

Bonnie B. Blaimer / ZooKeys 199: 23–70 (2012) 26 PTW Petiole width maximum width of petiole in dorsal view. PPL Postpetiole length measured in dorsal view at an angle that maximizes length. PPW Postpetiole width measured in same view as and perpendicular to postpeti- ole length. WL Weber’s length measured in lateral profile of mesosoma, distance from ap- proximate inflection point, where downward sloping pronotum curves into anteriorly projecting neck, to ventroposterior propodeal lobes. SPL Propodeal spine length measured from tip of propodeal spine to closest point on outer rim of propodeal spiracle, maximizing spine length in lateral view. LHT Length of metatibia, excluding the proximomedial condyle. CI Cephalic index: HW/HL. OI Ocular index: EL/HL. SI Scape index: SL/HW. PTHI Petiole height index: PTH/PTL. PTWI Petiole width index: PTW/PTL. PPI Postpetiole width index: PPW/PPL. SPI Propodeal spine index: SPL/WL. LBI Leg-body index: WL/LHT. Queen-specific measurements: MSNW Mesonotal width maximum width of mesonotum, measured in dorsal view. MSNL Mesonotal length maximum length of mesonotum, measured in dorsal view. MSNI Mesonotal index: MSNW/MSNL. Color images were created with a JVC KY-F75U digital camera, a Leica MZ16A stereomicroscope and ZERENE STACKER (v1.02) software. The scanning electron microscope images were taken at the California Academy of Sciences using a Zeiss/LEO 1450VP SEM. All ant images presented here are also publicly available on AntWeb (www.antweb.org). Line drawings were produced by tracing color images in Adobe Illus- trator CS5. Species distributions were plotted with ARCMAP (v9.3) within the software ARCGIS, based on coordinates (latitude and longitude) as given on the specimen labels of all material (see also supplementary table 1 for a species list with GPS coordinates). For material lacking this information, i.e. syntype specimens, the following sources were used to georeference collection sites: the GEOnet Names Server (National Geospatial- Intelligence Agency 2010) and the Gazetteer to Malagasy Botanical Collecting Locali- ties (Schatz and Lescot 2003). Classification of major geographic regions in Madagascar throughout species descriptions follows Gautier and Goodman (2003). Common abbre- viations within locality data are: P.N. = Parc National, R.S. = Réserve Spéciale, F = Forêt, P.C. = Parc Naturel Communautaire, R.N.I. = Réserve Naturelle Intégrale.

Taxonomy and species-groups of the subgenus Crematogaster (Orthocrema)... 27 The International Commission on Zoological Nomenclature (1999) requires lecto- types designated after 1999 to “contain an express statement of deliberate designation” (amended Article 74.7.3). I use the statement ‘lectotype by present designation’ to fulfill this requirement. Lectotypes have been designated where a name lacks a holotype or lec- totype and unambiguous syntypes have been identified. The purpose is to provide stabil- ity of nomenclature, and designation is done in a revisionary context in agreement with the amended Recommendation 74G of Article 74.7.3. Neotype designations have fur- ther been made for names with no extant name-bearing types that are in need of a name- bearing type “to objectively define the nominal taxon” (Article 75.1, ICZN, 1999), and are in agreement with the qualifying conditions stated in Article 75.3 (ICZN, 1999). Specimens were examined and/or deposited in the following collections: CASC California Academy of Sciences, San Francisco, CA, USA BBBC B.B. Blaimer Collection, University of California at Davis, CA, USA MCZC Museum of Comparative Zoology, Harvard, USA MHNG Muséum d’Histoire Naturelle, Genève, Switzerland MSNG Museo Civico di Storia Naturale, Genova, Italy NHMB Naturhistorisches Museum, Basel, Switzerland PSWC P.S. Ward Collection, University of California at Davis, CA, USA SAMC South African Museum, Cape Town, South Africa ZMBH Museum für Naturkunde der Humboldt Universität, Berlin, Germany Molecular data collection and phylogenetic analyses After sorting all available specimens to morphospecies, one to four individual worker ants for each of six putative Malagasy Crematogaster (Orthocrema) species were selected for genetic analysis. Four non-Malagasy Orthocrema species were chosen as outgroups, given their approximate relationships to the Malagasy taxa as known from a previous, larger phylogenetic analysis (Blaimer, in prep.). Two of these (C. sordidula Nylander and C. longipilosa Forel) represent distant relatives to all Malagasy Orthocrema, whereas the remaining two taxa (C. arcuata Forel and C. cf. dolens Forel) are closer relatives to the Malagasy taxa. For the distribution of the sampled taxa refer to Table 1. From these 17 specimens, DNA was extracted from either entire worker adults or pupae using a DNeasy Tissue Kit (Qiagen Inc., Valencia, California, U.S.A.), follow- ing the manufacturer’s protocol but eluting the extract in sterilized water rather than the supplied buffer and at half the suggested volume. I used either a non-destructive method (cuticle of ant pierced prior to extraction, mostly used for adults), enabling me to re- tain and re-mount voucher specimens after extractions, or a destructive technique (en- tire ant pulverized, mostly used for pupae) in cases where multiple individuals from the same colony series were available. Three nuclear protein-coding genes were amplified: long wavelength rhodopsin (LW Rh, 856bp exon /255bp intron), arginine kinase (ArgK,