Corresponding author: Bernhard Seifert (
Academic editor: Brian Fisher
The paper integrates two independent studies of numeric morphology-based alpha-taxonomy of the cryptic ant species
Seifert B, Csösz S (2015)
The small Formicoxenine ants
According to mtDNA data (
Frequent hybridization of
Very early it became obvious to the senior author (Seifert) that there existed a third cryptic entity of the
Morphology is essential to establish the link between species delimitation and Linnean nomenclature (
In this paper, we firstly present our argumentation why
A total of 203 nest samples with 603 individual workers of both species was investigated – 104 samples by Seifert and 99 by Csösz. Only one nest series, the type series of
A total of 119 nest samples originated from the following countries: Austria 13, Bosnia 2, Bulgaria 7, Czechia 3, Germany 39, Greece 24, Hungary 8, Italy 2, Macedonia 3, Moldova 2, Poland 5, Serbia 1, Slovenia 4 and Ukraine 7 samples. A detailed account of the samples is given in the following under the sequence site, date in the format yyyy.mm.dd, sample No [latitude in decimal format, longitude in decimal format, altitude].
A total of 84 nest samples originated from the following countries: Armenia 1, Bulgaria 31, Georgia 6, Greece 15, Romania 4, Russia 2, Turkey 21 and Ukraine 4 samples. A detailed account of the samples is given in the following.
The type series, stored in Shmalhausen Institute of Zoology Kiev and certainly representing a nest sample, is labeled “Kiev: Golosejev (Karavaijev No 3057)”. Three syntype workers were investigated by Seifert and seven syntype workers by Csösz.
The paratypes, seven workers on the same pin and originating from the nest that contained the queen holotype, are labeled “Kr. Görlitz, 19.3.1993, Schönau-Berzdorf, Hutberg, g31”, „
The worker holotype is labelled “BUL:
The senior and junior author performed two independent investigations of worker ant morphology, considering different character combinations and using different microscopic equipment. Seifert recorded 18 and Csösz 22 primary morphometric characters. In bilaterally developed characters, arithmetic means of both body sides were calculated. All measurements were made on mounted and fully dried specimens. Measurements of body parts always refer to real cuticular surface and not to the diffuse pubescence surface.
Seifert used for spatial adjustment of specimens a pin-holding stage, permitting full rotations around X, Y, and Z axes and a Leica M165C high-performance stereomicroscope equipped with a 2.0 planapochromatic objective (resolution 1050 lines/mm) at magnifications of 120–384×. The mean relative measuring error over all magnifications was 0.3%. A Schott KL 1500 cold-light source equipped with two flexible, focally mounted light-cables, providing 30°-inclined light from variable directions, allowed sufficient illumination over the full magnification range and a clear visualization of silhouette lines. A Schott KL 2500 LCD cold-light source in combination with a Leica coaxial polarized-light illuminator provided optimal resolution of tiny structures and microsculpture at highest magnifications. Simultaneous or alternative use of the cold-light sources depending upon the required illumination regime was quickly provided by regulating voltage up and down. A Leica cross-scaled ocular micrometer with 120 graduation marks ranging over 52% of the visual field was used. To avoid the parallax error, its measuring line was constantly kept vertical within the visual field.
Equipment and measurement procedures of Csösz
Measurements were made using a pin-holding stage, permitting rotations around X, Y, and Z axes. An Olympus SZX9 stereomicroscope was used at a magnification of 150× for each character, allowing a precision of ± 2 µm.
29 morphometric characters were investigated – ten of these by both authors. We give the character definitions in alphabetic order. In square brackets is indicated who investigated a character.
Explanation of morphometric characters.
The delimitation of the cryptic species was done by an interaction of Nest-Centroid Clustering (NC clustering) and a confirmative linear discriminant analysis (LDA). NC Clustering was run both as hierarchical NC-Ward clustering and non-hierarchical NC-K-means clustering. These methods were described in more detail by
The same mathematical procedures were applied for the data sets of Seifert and Csösz. NC-Ward clustering was run first to indicate the putative number of K main clusters. In the second step NC-K-Means was performed with the setting of K classes suggested by NC-Ward. Classifications being coincident between the hierarchical and non-hierarchical clustering formed the hypothesis for the controlling LDA that was subsequently run. Samples with classifications disagreeing between NC-Ward and NC-K-means were run in this LDA as wild-cards. The final classification (“final species hypothesis”) was established by the LDA in the iterative procedure described by
The W Palaearctic species of the
Head short, mean index CL/CW only 1.053–1.063.
Whole dorsum of vertex regularly and continuously longitudinaly carinulate, shining surface areas are absent or restricted to a narrow median stripe.
Metanotal depression always visible, at least suggested.
Antennal club and femora never with a blackish pigmentation
Petiole in lateral view rather high and with a weakly concave frontal face; the anterior profiles of node and peduncle form an angle of about 150–155° whereas anterior and dorsal profiles of node form an angle of 90–105°. Dorsal profile of node steeply sloping down to caudal cylinder. The profiles of this slope and of the caudal cylinder form an angle of about 140°.
Propodeal spines acute, deviating from longitudinal axis of mesosoma by 32–42° and moderately long, SP/CS 0.200–0.260, SPST/CS 0.253–0.356.
In the data set of Seifert and considering all 18 characters, NC Ward clustering provided a clear separation of the cryptic species in two main branches (Fig.
The results were similar in the data set of Csösz, considering all 22 characters and following the same procedure. The classifications of NC-Ward and NC-K-means differed in 11.1% of samples. NC-Ward disagreed from the final species hypothesis in 11.1% and NC-K-means in 3.1% of samples. A character reduction by a stepwise LDA, again performed iteratively, improved the classification result significantly. NC-Ward disagreed from the final species hypothesis in only 2.1% (Fig.
We consider the congruent results of two independent investigations and investigators and the low disagreement of 1.1–2.1% between the classifications of exploratory data analyses with the final species hypothesis as a strong argument for heterospecificity of
We achieved an agreement of 97.9–98.9% between the final species hypothesis and four different cluster analyses. This indicates a significant evolutionary divergence and could indicate that no or very few hybrids are present in our data set. Yet, the present problem is more difficult because extremely similar species are involved which prevents a reliable phenotypic identification of a particular hybrid sample. A broader statistic approach is needed. Accordingly, we compare the positions of samples along the interspecific LDA vector (i) from the potential contact zone, (ii) from the allopatric zones and (iii) from sites with established syntopic occurrence of both species. Reduced interspecific distances in contact zones are an indication that hybridization could have occurred. We consider the territories of Greece, Bulgaria, Romania and Moldova to form the potential contact zone (Fig.
There is only a very weak, insignificant reduction of the distance from the zero point of the discriminant vector in samples from the potential contact zone compared to those from the allopatric zones (Tab.
Distance of the sample means of
Distance from zero | |
---|---|
Potential contact zone (total 84, |
1.6173 ± 1.0000 [0.1186.3.8912] |
ANOVA [F, p] | 0.370, 0.543 |
Allopatric zones (total 119, |
1.6941 ± 0.7906 [0.0326,3.4717] |
ANOVA [F, p] | 11.267, 0.001 |
Syntopic sites (total 31, |
1.1465 ± 0.8740 [0.1186,3.4894] |
ANOVA [F, p] | 12.383, 0.001 |
Potential contact zone without syntopic sites (total 53, |
1.8927 ± 0.9727 [0.1258,3.8912] |
We conclude that
Seifert proposed as operational criterion for the discipline “multivariate investigation of ant worker morphology” and as a remedy against over-splitting that at least 97% of the classifications by exploratory data analyses should agree with the classifications by linear discriminant analyses that form the final species hypothesis. However, 4% disagreement or 96% confirmation seem to be a more reasonable threshold when considering the performance of the two most powerful methods of NC-clustering, NC-K-Means and NC-Ward, in the separation of 74 cryptic ant species (Table 1 in
The consequences of threshold decisions may be illustrated by an example. Over many years we studied the situation in the Mediterranean
We want to emphasize in this context that hybridization and reticulate evolution is a matter of fact in the living nature around us (reviewed, e.g., in Abbott et al. 2013). Exemplary groups in animals are ducks (
Finally we address the question if our two entities might represent an intraspecific dimorphism instead of representing different species by considering geographic distribution and the frequency and distribution of supposedly mixed nests in the sympatric and allopatric zones. Frequencies of discrete morphs provided by the gene pool of a single species may show steep geographic gradients - remind alone of the famous text book example of the Peppered Moth
In the provisional internal naming system of the senior author the new species had been designated over the years as “
See above under “Material”.
All morphometric data given in the following verbal description are arithmetic means of 256 worker individuals calculated by fusing Seifert’s and Csösz’s data sets. Harmonization of the different data sets has been performed by the function CW = 1.0791 * CWb.
Data set of Seifert; nest sample means of morphometric data of the workers of the cryptic species
ANOVA |
|||
---|---|---|---|
CS | 641 ± 36 [594,713] | 0.02, n.s. | 645 ± 30 [556,713] |
CL/CW | 1.065 ± 0.014 [1.038,1.087] | 16.90, 0.000 | 1.053 ± 0.013 [1.013,1.081] |
SL/CS | 0.767 ± 0.011 [0.741,0.786] | 1.38, n.s. | 0.764 ± 0.013 [0.734,0.790] |
PoOc/CL | 0.395 ± 0.007 [0.380,0.406] | 3.63, n.s. | 0.392 ± 0.007 [0.373,0.405] |
EYE/CS | 0.210 ± 0.005 [0.201,0.219] | 29.22, 0.000 | 0.216 ± 0.005 [0.207,0.233] |
FRS/CS | 0.361 ± 0.008 [0.344,0.376] | 2.48, n.s. | 0.364 ± 0.008 [0.345,0.386] |
SPBA/CS | 0.280 ± 0.010 [0.252,0.297] | 26.53, 0.000 | 0.294 ± 0.012 [0.268,0.322] |
SPTI/CS | 0.325 ± 0.014 [0.295,0.347] | 0.350 ± 0.013 [0.324,0.377] | |
SPST/CS | 0.285 ± 0.014 [0.250,0.308] | 0.323 ± 0.014 [0.286,0.354] | |
SP/CS | 0.218 ± 0.015 [0.188,0.246] | 0.260 ± 0.012 [0.232,0.290] | |
PEW/CS | 0.253 ± 0.007 [0.242,0.270] | 6.76, 0.011 | 0.258 ± 0.008 [0.239,0.275] |
PPW/CS | 0.353 ± 0.010 [0.335,0.375] | 24.77, 0.000 | 0.367 ± 0.013 [0.331,0.395] |
PEH/CS | 0.357 ± 0.008 [0.343,0.374] | 14.22, 0.000 | 0.364 ± 0.009 [0.343,0.391] |
PEL/CS | 0.472 ± 0.011 [0.452,0.497] | 7.41, 0.008 | 0.479 ± 0.013 [0.446,0.508] |
ML/CS | 1.187 ± 0.016 [1.158,1.221] | 0.08, n.s. | 1.188 ± 0.015 [1.156,1.239] |
MW/CS | 0.605 ± 0.010 [0.580,0.628] | 3.68, n.s. | 0.600 ± 0.011 [0.780,0.626] |
MH/CS | 0.524 ± 0.011 [0.500,0.547] | 11.93, 0.001 | 0.532 ± 0.012 [0.507,0.566] |
MPGR/CS | 2.10 ± 0.56 [1.31,3.41] | 0.25, n.s. | 2.04 ± 0.49 [1.07,3.23] |
Data set of Csösz; nest sample means of morphometric data of the workers of the cryptic species
ANOVA F, p | |||
---|---|---|---|
CSb | 614 ± 37 [539,718] | 1.73, n.s. | 623 ± 36 [544,688] |
CL/CWb | 1.155 ± 0.018 [1.120,1.196] | 14.03, 0.000 | 1.140 ± 0.021 [1.082,1.180] |
SL/CSb | 0.791 ± 0.013 [0.763,0.836] | 6.40, 0.013 | 0.784 ± 0.013 [0.756,0.811] |
PoOc/CL | 0.390 ± 0.007 [0.379,0.405] | 0.00, n.s. | 0.390 ± 0.007 [0.370,0.410] |
EL/CSb | 0.255 ± 0.007 [0.236,0.279] | 0.71, n.s. | 0.256 ± 0.005 [0.247,0.268] |
FRS/CSb | 0.375 ± 0.008 [0.358,0.397] | 1.57, n.s. | 0.377 ± 0.008 [0.362,0.400] |
MW/CSb | 0.630 ± 0.010 [0.610,0.662] | 1.37, n.s. | 0.627 ± 0.017 [0.590,0.678] |
ML/CSb | 1.227 ± 0.017 [1.193,1.268] | 1.01, n.s. | 1.223 ± 0.023 [1.183,1.303] |
SPBA/CSb | 0.298 ± 0.012 [0.277,0.321] | 32.89, 0.000 | 0.312 ± 0.013 [0.282,0.339] |
SPTI/CSb | 0.333 ± 0.016 [0.300,0.384] | 0.365 ± 0.016 [0.332,0.397] | |
SPWI/CSb | 0.352 ± 0.018 [0.295,0.405] | 0.388 ± 0.016 [0.355,0.422] | |
SPST/CSb | 0.289 ± 0.015 [0.253,0.319] | 0.330 ± 0.011 [0.298,0.356] | |
SPL/CSb | 0.162 ± 0.006 [0.149,0.175] | 17.06, 0.000 | 0.157 ± 0.008 [0.137,0.175] |
PEW/CSb | 0.269 ± 0.008 [0.252,0.288] | 10.18, 0.002 | 0.275 ± 0.011 [0.255,0.317] |
PEH/CSb | 0.372 ± 0.009 [0.354,0.398] | 7.88, 0.006 | 0.377 ± 0.009 [0.361,0.400] |
NOH/CSb | 0.169 ± 0.007 [0.156,0.202] | 29.11, 0.000 | 0.177 ± 0.007 [0.164,0.189] |
NOL/CSb | 0.254 ± 0.010 [0.232,0.274] | 4.68, 0.033 | 0.258 ± 0.008 [0.237,0.275] |
NODL/CSb | 0.296 ± 0.014 [0.266,0.318] | 0.02, n.s. | 0.295 ± 0.017 [0.267,0.340] |
PPW/CSb | 0.368 ± 0.009 [0.348,0.387] | 19.17, 0.000 | 0.377 ± 0.012 [0.348,0.405] |
PPH/CSb | 0.350 ± 0.008 [0.335,0.368] | 7.74, 0.007 | 0.356 ± 0.011 [0.340,0.384] |
PPL/CSb | 0.255 ± 0.009 [0.234,0.273] | 1.04, n.s. | 0.257 ± 0.008 [0.236,0.272] |
PL/CSb [%] | 0.413 ± 0.011 [0.383,0.434] | 5.74, 0.019 | 0.419 ± 0.013 [0.393,0.454] |
Mesosoma moderately wide (MW/CS 0.608) and metanotal depression always developed (MpGr/CS 2.1%). Propodeal spines rather long and acute but distinctly shorter than in
Petiole in lateral view rather high and with a weakly concave frontal face; the anterior profiles of node and peduncle form an angle of about 150–155° whereas anterior and dorsal profiles of node form an angle of 90–105°. Dorsal profile of node weakly convex or nearly straight and moderately long, steeply sloping down to caudal cylinder. The profiles of this slope and of the caudal cylinder form an angle of about 140°. Whole surface of petiolar and postpetiolar nodes microreticulate with a mesh width of 9–13 µm. Two longitudinal rugae typically demarcate the margin of dorsal petiolar plane while the sides of petiolar tergites are stabilized by one longitudinal carina/ruga on each side.
Overall body color dirty yellow to light brown with a strong yellowish component. Mesosoma, appendages, waist and basis of first gaster tergite usually lighter yellow to dirty yellow. Head dorsum and the posterior surfaces of gaster tergites usually darker, generally yellowish brown. Lighter heads occur.
There is considerable overlap in each of the 29 shape characters and absolute size (Tables
D(6) = 22.058*PoOc+17.640*SL-66.166*SPST+38.233*MW-28.926*PPW -35.873*SPTI-1.797
classified 203 nest samples with an error of 3.4%. Samples with an arithmetic mean of D(6) < 0 are determined as
The most simple means for separation of
D(3) = 129.53*SPBA–120.88*PPW+133.8*MpGR +4.446
classified 87 nest samples with an error of 3.4%. Samples with an arithmetic mean of D(3) < 0.64 are determined as
The present zoogeography (Fig.
We wish to thank Vera Antonova / Sofia and Andreas Schulz / Leverkusen for providing a lot of samples from the Balkans and Asia Minor, Alexandr Radchenko / Kiev for enabling a loan of type specimens of