Taxonomy of Malagasy Nesomyrmex brevicornis species-group using conventional morphology-based approach

Abstract Here we describe the diversity of the Malagasy Nesomyrmex brevicornis-group as the final installment of series describing the complete Malagasy Nesomyrmex fauna. In the current paper introduce the Nesomyrmex brevicornis-group, a newly outlined species group. We split this group from one of the four former groups, Nesomyrmex madecassus-group (sensu Csősz and Fisher 2015). The relatively small number of individuals available within the Nesomyrmex brevicornis-group relative to the large number of OTUs hampers the use of sophisticated statistical techniques to evaluate the morphological data. Instead, we assess the diversity of the Nesomyrmex brevicornis-group via a conventional morphology-based approach relying upon non-numeric characters (shape, sculpture, color etc.) recorded by simple eye inspection. Morphometric data are used as secondary data to confirm the most probable boundaries between species, but without employing statistical analyses. We rejected prior species hypotheses imposed by the primary approach if a non-overlapping range of a single index calculated from continuous numeric traits was found between two hypothesized species. Our methods render every species in this revisionary work separable via both salient features and morphometric traits. Our approach reveals the existence of nine morphologically distinct species, Nesomyrmex brevicornis sp. n., Nesomyrmex brunneus sp. n., Nesomyrmex cingulatus sp. n., Nesomyrmex edentates sp. n., Nesomyrmex flavigaster sp. n., Nesomyrmex longiceps sp. n., Nesomyrmex minutus sp. n., Nesomyrmex punctaticeps sp. n., and Nesomyrmex sellaris sp. n.. Geographic maps showing the distribution of each species are provided, along with an updated key to workers of Malagasy Nesomyrmex species groups that includes the newly outlined Nesomyrmex brevicornis-group.


Introduction
The ant genus Nesomyrmex has been the subject of intensive systematic research in recent years, and contributes to efforts to explore the ant fauna of the Malagasy zoogeographical region, i.e. Madagascar and surrounding islands (Bolton 1994). Thanks to earlier explorations of the region's biodiversity, our knowledge of the Malagasy Nesomyrmex fauna has increased considerably. The Malagasy members of the genus Nesomyrmex were split into four remarkable species groups by Csősz and Fisher (2015): N. angulatus-group (Csősz and Fisher 2016a), N. hafahafa-group (Csősz and Fisher 2015), N. madecassus-group (Csősz and Fisher 2016c) and N. sikorai-group (Csősz and Fisher 2015). Their classifications were revised using combined algorithmic approaches to evaluate numeric morphological data. The total species number has climbed to 24 of which 20 are new to science, underpinning earlier assumptions about the extreme species richness of the region.
In the current paper we complete the reexamination of Malagasy Nesomyrmex with a revision of the fifth species group, N. brevicornis-group, and by describing the nine species that are new to science. This group is subsequently split from the N. madecassus-group (sensu Csősz and Fisher 2015) based on several features, such as the remarkably deep mesopropodeal depression, the short scape (SL/CS: 0.49-0.72, in contrast to SL/CS of madecassus-group species: 0.72-0.83), the short and high petiole (PEL/ CS: 0.34-0.49, in contrast to that of madecassus-group species: 0.44-0.58), the relatively long petiolar node (NOL/CS: 0.20-0.34, in contrast to that of madecassus-group species: 0.28-0.39, and the shorter mesosoma (ML/CS: 1.08-1.36, in contrast to that of madecassus-group species: 1.26-1.50) that distinguish workers of brevicornis-group from those of madecassus-group species.
All species of the bervicornis-group are arboreal; most nest in twigs or hollow branches, but some have very specialized associations with live stems or particular species of trees. Nesomyrmex cingulatus sp. n., for example, is known only from live stems from spiny thicket near Tsimanampetsotsa. N. minutus sp. n. is unique in nesting only in the stems of Macphersonia gracilis (Sapindaceae) and represents one of the few known ant plants in Madagascar. The specialized nesting habits of the bervicornisgroup may explain why collections of this group are minimal compared to other Nesomyrmex species.
As the material available within this species group is insufficiently large for statistical evaluation, morphological diversity is assessed via evaluation of qualitative traits or trait-combination. Species are believed to have characteristic shapes, sizes, pigmentation patterns, sculpture, and other external features that may help in species separation and subsequent identification (Strauss and Bond 1990), hence qualitative morphological traits have often been the primary source for taxonomic studies (see e.g. Hita- Garcia and Fisher 2014, Snelling et al. 2014, Prebus 2015). An obvious advantage of this approach that it does not require large sample sizes to set species hypotheses. However, the often smaller number of available specimens hampers statistically supported hypothesis testing, and without morphometric analyses cryptic taxa may re-main undiscovered. Overall, conventional morphology-based taxonomy remains the primary approach to outline species boundaries when small sample size precludes more complex research.
Qualitative evaluation of morphological data has revealed the existence of nine morphologically distinct species within N. brevicornis species-group in the Malagasy zoogeographical region, Nesomyrmex brevicornis sp. n., N. brunneus sp. n., N. cingulatus sp. n., N. edentates sp. n., N. flavigaster sp. n., N. longiceps sp. n., N. minutus sp. n., N. punctaticeps sp. n., and N. sellaris sp. n.. We provide a traditional, character-based key supported by simple numeric data based on body size ratios. Geographic maps showing the distribution of these species where populations of particular species were collected are also provided.

Material and methods
In the present study, 21 continuous morphometric traits were recorded in 146 worker individuals belonging to 111 nest samples and 35 further single individuals collected in the Malagasy region.
The material is deposited in the following institutions, abbreviations after Evenhuis (2013): CASC (California Academy of Sciences, San Francisco, California, U.S.A.), MCZC (Museum of Comparative Zoology, Cambridge, Massachusetts, U.S.A.) and PSWC (Phil S. Ward's collection, University of California Davis, Davis, California, U.S.A.). Type material and samples that were morphometrically investigated is presented in "Type material investigated" and "Material examined" sections in the format as follows: CASENT code, collection code, verbatim locality, longitude, latitude, elevation in meter, collector, date in MM.DD.YYYY format, (gender, abbreviation of depository). For genders (queen, worker, male) the first letter (Q, w, m) is used.
All images of specimens used in this study are available online on AntWeb (http:// www.antweb.org). Images are linked to their specimens via the unique specimen code affixed to each pin (CASENT0027494). Online specimen identifiers follow this format: http://www.antweb.org/specimen/CASENT0027494. Digital color montage images were created using a JVC KY-F75 digital camera and Syncroscopy Auto-Montage software (version 5.0), or a Leica DFC 425 camera in combination with the Leica Application Suite software (version 3.8). Distribution maps were generated in R (R Core Team 2015) via 'phylo.to.map' function using package phytools (Revell 2012).
Measurements were taken with a Leica MZ 12.5 stereomicroscope equipped with an ocular micrometer at a magnification of 100×. Measurements and indices are presented as arithmetic means with minimum and maximum values in parentheses. Body size dimensions are expressed in µm. Due to the abundance of worker individuals available relative to queen and male specimens, the present revision is based on worker caste only. Worker-based revision is further facilitated by the fact that the name-bearing type specimens of the vast majority of existing ant taxa belong to the worker caste. All measurements were made by the first author. For the definition of morphometric characters, earlier protocols (Csősz et al. 2015, Csősz and Fisher 2015, 2016a, 2016b were considered. Explanations and abbreviations for measured characters are as follows:

CL
Maximum cephalic length in median line. The head must be carefully tilted to the position providing the true maximum. Excavations of hind vertex and/or clypeus reduce CL.

CW
Maximum width of the head. Includes compound eyes.

CWb
Maximum width of head capsule without the compound eyes. Measured just posterior of the eyes.

CS
Absolute cephalic size. The arithmetic mean of CL and CWb.

Cdep
Antero-median clypeal depression. Maximum depth of the median clypeal depression on its anterior contour line as it appears in fronto-dorsal view.

EL
Maximum diameter of the compound eye.

FRS
Frontal carina distance. Distance of the frontal carinae immediately caudal of the posterior intersection points between frontal carinae and the torular lamellae. If these dorsal lamellae do not laterally surpass the frontal carinae, the deepest point of scape corner pits may be taken as the reference line. These pits occupy the inner corner of the scape base when the scape is directed caudally and produce a dark triangular shadow in the lateral frontal lobes immediately posterior to the dorsal lamellae of the scape joint capsule.

ML (Weber length)
Mesosoma length from caudalmost point of propodeal lobe to transition point between anterior pronotal slope and anterior pronotal shield. Preferentially measured in lateral view; if the transition point is not well defined, use dorsal view and take the center of the dark-shaded borderline between pronotal slope and pronotal shield as anterior reference point. In gynes: length from caudalmost point of propodeal lobe to the most distant point of steep anterior pronotal face.

MW
Mesosoma width. In workers MW is defined as the longest width of the pronotum in dorsal view excluding the pronotal spines. MPST Maximum distance from the center of the propodeal stigma to the anteroventral corner of the ventrolateral margin of the metapleuron. NOH maximum height of the petiolar node. Measured in lateral view from the uppermost point of the petiolar node perpendicular to a reference line set from the petiolar spiracle to the imaginary midpoint of the transition between dorso-caudal slope and dorsal profile of caudal cylinder of the petiole.

NOL
Length of the petiolar node. Measured in lateral view from the center of petiolar spiracle to dorso-caudal corner of caudal cylinder. Do not erroneously take as the reference point the dorso-caudal corner of the helcium, which is sometimes visible.
PEH maximum petiole height. The chord of the ventral petiolar profile at node level is the reference line perpendicular to which the maximum height of petiole is measured.

PEL
Diagonal petiolar length in lateral view; measured from anterior corner of subpetiolar process to dorso-caudal corner of caudal cylinder.

PEW
Maximum width of petiole in dorsal view. Nodal spines are not considered. PoOC Postocular distance. Use a cross-scaled ocular micrometer and adjust the head to the measuring position of CL. Caudal measuring point: median occipital margin; frontal measuring point: median head at the level of the posterior eye margin.

PPH
Maximum height of the postpetiole in lateral view. Measured perpendicularly to a line defined by the linear section of the segment border between dorsal and ventral petiolar sclerite.

PPL
Postpetiole length. The longest anatomical line that is perpendicular to the posterior margin of the postpetiole and is between the posterior postpetiolar margin and the anterior postpetiolar margin.

PPW
Postpetiole width. Maximum width of postpetiole in dorsal view.

SL
Scape length. Maximum straight line scape length excluding the articular condyle.

SPST
Spine length. Distance between the center of propodeal stigma and spine tip. The stigma center refers to the midpoint defined by the outer cuticular ring but not to the center of the real stigma opening, which may be positioned eccentrically.

SPTI
Apical spine distance. The distance of spine tips in dorsal view; if spine tips are rounded or truncated, the centers of spine tips are taken as reference points.
In verbal descriptions of taxa based on external morphological traits, recent taxonomic papers Fisher 2015, 2016) were considered. Definitions of surface sculpturing are linked to Harris (1979). Body size is given in µm, and means of morphometric ratios as well as minimum and maximum values are given in parentheses with up to three digits. Inclination of pilosity and cuticular spines is given in degrees. Definitions of species-groups as well as descriptions of species are surveyed in alphabetic order.
Hypothesis formation. The present paper aims to reveal diversity of N. brevicornis species-group, including a number of very rare species represented only by three to four individuals. This high level of asymmetry in data, coupled with the extremely low average number of individuals per species, prevents employment of statistical analyses in hypothesis formation and testing. Instead, the conventional morphology-based approach (i.e., individuals are sorted into clusters using simple eye inspection based on salient morphological features, e.g., shape, sculpture etc.) is used as the primary technique for recognizing species. Morphometric data are given for each species as secondary data for confirmation and in order to improve identification.

Nesomyrmex cingulatus
Etymology. The name (flavigaster) refers to the light yellow gaster of this species, which is in sharp contrast to the dark brown color of the rest of the body.