Myrmecological News 23 41-59 Vienna, September 2016

Analyzing large-scale and intranidal phenotype distributions in eusocial Hymenoptera – a taxonomic tool to distinguish intraspecific dimorphism from heterospecificity

Bernhard SEIFERT

Abstract Ant and termite nests are long-term stable, semi-closed systems constantly producing conspecific worker populations of related individuals over many generations. Accordingly, nests of these eusocial insects, as they are found in nature, offer free of cost an analysis situation that has to be generated in other groups of organisms by controlled rearing experiments. A test system based on analyzing intranidal and large-scale phenotype distributions and comparing the observed distributions with predictions for different scenarios of heterospecificity and intraspecific dimorphism is introduced by a case study on ants. The test system, named DIMORPH test, allows a taxonomist to distinguish if discrete character syndromes represent separate species or an intraspecific phenomenon. One of the most important parameters within the test system is the abundance and distribution of phenotypically mixed nest populations. Five biological explanations are possible for ant nests with a mixture of discrete phenotypes: They may represent (1) geneti- cally determined intraspecific morphs, (2) intraspecific modifications induced by environmental factors, (3) the associ- ation of a temporary social parasite with a host species, (4) the association of a permanent social parasite with a host species, and (5) a parabiotic association of two basically independent (self-sustaining) species. The paper explains the biological background of the scenarios (1) to (5) and presents mathematical models and generalizations from empirical data to predict phenotype distributions for each scenario under variable conditions. Four cases of intraspecific dimorphism and five cases of taxonomically recognized pairs of cryptic or similar species are presented and analyzed. The observed intranidal phenotype distribution was most similar to the predicted scenario of intraspecific dimorphism in Camponotus lateralis (OLIVIER , 1792), Lasius umbratus (NYLANDER , 1846), Formica lugubris ZETTERSTEDT , 1838, and Cardiocondyla elegans EMERY , 1869. In three of these examples, intraspecific morphs had been considered previously as different species. Heterospecificity was confirmed for four pairs of cryptic species and one pair of closely related species: Formica pressilabris NYLANDER , 1846 vs. F. foreli BONDROIT , 1918, Temnothorax crassispinus (KARAVAJEV , 1926) vs. T. cra- secundus SEIFERT & CSÖSZ , 2015, Temnothorax luteus (FOREL , 1874) vs. T. racovitzai (BONDROIT , 1918), two cryptic species of the Pheidole pallidula (NYLANDER , 1849) complex, and Myrmica vandeli BONDROIT , 1920 vs. M. scabrinodis NYLANDER , 1846. The phenotype-based DIMORPH test can be applied to the large worldwide collections of mounted museum material or private collections of ants independent from age or DNA degradation and can thus operate in fields where genetic investigation faces analytical and logistic problems and where controlled rearing experiments are not possible. The system can be adapted, with some modification, to other groups of eusocial organisms. Key words: Numeric morphology-based alpha-taxonomy, pleiotropy, intraspecific polymorphism, parabiosis, temporary social parasitism, permanent social parasitism. Myrmecol. News 23: 41-59 (online 21 April 2016) ISSN 1994-4136 (print), ISSN 1997-3500 (online) Received 16 November 2015; revision received 11 January 2016; accepted 13 January 2016 Subject Editor: Heikki Helanterä Bernhard Seifert , Senckenberg Museum für Naturkunde Görlitz , Am Museum 1 , 02826 Görlitz , Germany. E-mail: [email protected]

Introduction There is some automatism for taxonomists to suppose he- based taxonomists cannot a priori fulfill this requirement terospecificity for clearly separable phenotypes when they when studying a group of organisms living solitarily, in show a degree and type of differences as they are usual temporary pairs or in random instable associations such between species in the group of organisms under study. as swarms of fishes or herds of antelopes. A taxonomist Such expectations will prove true in the majority of cases. cannot a priori distinguish between heterospecificity and Yet, the task of taxonomists is not finished at this stage. intraspecific dimorphism if there are a number of more They should develop a sense for the unexpected, and some- thickset fishes with more greenish iridescence and more times the unexpected is not the very exception. The Prag- pointed pectoral fins in a swarm of normal herrings. In such matic Species Concept (SEIFERT 2014) recommends as a groups of organisms, the decision process essentially needs taxonomic working routine to check if distinct phenotypes information from sources other than morphology. These could represent an intraspecific polymorphism. Morphology- are in the best case a combination of nuDNA genetics with long-term observation of mating and reproduction beha- textbook examples are Panthera melas (CUVIER , 1809) vior in nature or in laboratory. which is a taxonomic name for the black mutant of the Eusocial insects, in contrast, are an ideal object for Leopard Panthera pardus (LINNAEUS , 1758) or Araschnia taxonomic a priori assessment. They provide sources of in- prorsa (LINNAEUS , 1758) that is a name for an environment- formation not given in other groups of organisms. Nests induced modification of the butterfly Araschnia levana of ants, as they are found in nature, offer "free of charge" (LINNAEUS , 1758). an analysis situation that has to be generated in other groups The largest risks for taxonomists are presented by sys- of organisms by controlled rearing experiments. That is, tems of correlated, discrete characters which occur as a ant nests are long-term stable, semi-closed systems cons- syndrome. This can be explained by an example. There is tantly producing conspecific worker populations of related a Mediterranean Camponotus MAYR , 1861 carpenter ant individuals over many generations. Furthermore, collect- in which profuse scape pilosity, more linear frontal cari- ing workers in sufficient numbers is possible at any time nae, a weakly developed scape base extension, a shorter of the field season. If clean nest samples with several work- scape, a shorter propodeum and a broader head occur in a ers per nest are collected and if these samples are avail- fixed combination and there is another, apparently closely able from a reasonably large geographic area, a particular related Camponotus ant in which missing scape pilosity, analysis becomes possible. The taxonomist can analyse sinusoidally formed frontal carinae, a strongly developed within-nest and large-scale phenotype distribution and can scape base extension, a longer scape, a longer propodeum compare the observed distribution with predictions for dif- and a broader head occur in another fixed combination ferent scenarios of heterospecificity and intraspecific di- (Figs. 1, 2). Discrete phenotypes formed as a syndrome morphism. of multiple, seemingly independent characters should re- These analyses are introduced in this paper. They aim quire mutually compatible changes in many different gene to prevent taxonomists from misinterpreting discrete intra- loci and each ant taxonomist with some experience would specific character syndromes as separate species. This goal immediately assume here two clearly evolved species. This of distinguishing intraspecific dimorphism from hetero- conclusion was drawn in this case by three different au- specificity is realistic when the taxonomist has access to a thors: In the determination key of SEIFERT (2007) the hairy sufficiently large number of samples from a sympatric area. form was separated from Camponotus lateralis (OLIVIER , Beyond that, the paper wants to develop a sense among ant 1792) under the provisional designation " Camponotus la- taxonomists which scenarios have to be considered but it teralis sp. 2" and BOROWIEC & SALATA (2014) ascribed will not claim that the precise nature of a heterospecific the hairy form of C. lateralis to the species C. honaziensis intranidal association could be determined by analyses of KARAMAN & AKTAÇ , 2013 (see also the pictures coded within-nest and overall phenotype distributions alone. The CASENT0914262 in www.antweb.org). This paper will analyses and considerations of biological backgrounds pre- show, among other similar examples, that the two pheno- sented here refer to ants but are likely to be translated with types are provided by the same gene pool and represent some modification to other groups of eusocial organisms. an example of a treacherous intraspecific dimorphism of the ant C. lateralis. Background knowledge and the development of pre- This result may appear surprising for a taxonomist with dictions a view centered on morphology. Discrete character syn- The taxonomists' view on syndromes of discrete charac- dromes provided by the same gene pool are preferentially ters and their supposed genetic background: The danger explained by the pleiotropic action of a single regulatory of a taxonomic misinterpretation of intraspecific pheno- gene encoding a transcription factor. The genetic litera- type polymorphism varies with the form in which it oc- ture provides a lot of examples for monogenic regulation curs. If we consider syndromes of multiple characters and of character syndromes (e.g., GRUENEBERG 1938, JERKA - not a single character – such as a white, pink and red color DZIADOSZ & al. 1992, MCKONE & al. 2003, MARCELLINI of Gregor Mendel’s flowers which represent three dis- & SIMPSON 2006, PRUD 'HOMME & al. 2006, MCGREGOR crete phenotypes – intraspecific polymorphism based on co- & al. 2007, PRUD 'HOMME & al. 2007, WAYNE & OSTRAN - dominant inheritance of phenotypic characters does not DER 2007, STANKE & al. 2013, SCHWITZGEBEL 2014). In present a serious problem. The high frequency of inter- ants, monogenic dominant-recessive control of gyne poly- mediate forms in multi-character cases and the poorly de- morphism has been credibly shown in long-term rearing fined borders between phenotypes in attempts to cluster experiments by WINTER & BUSCHINGER (1986) for the these by exploratory and hypothesis driven analyses will slave-hunting ant Harpagoxenus sublaevis (NYLANDER , reduce the probability of considering the homozygous geno- 1849) and by BUSCHINGER (2005) for Myrmecina grami- types as different species. More dangerous are discrete nicola (LATREILLE , 1802). A similar mechanism was sug- characters controlled by dominant-recessive inheritance or gested for a Nearctic Leptothorax MAYR , 1855 species other mechanisms. If there is a discrete difference in only (HEINZE & BUSCHINGER 1987). a single character, such as a simple dark / light color dif- Discrete character syndromes may also be controlled ference, cautious taxonomists more readily consider the by the combination of several genes with low crossing- possibility of intraspecific dimorphism. They are usually over frequency in a linkage group on the same chromo- aware that a single point mutation may cause such a change some. This "supergenic" mechanism is supposed to be rarer as shown, for example, by LUS (1932), BARRION & SAXENA than monogenic control. However, such cases have been (1987), MAJERUS (1998), ANDRES & CORDERO (1999), documented recently for ants and are possibly more fre- MAJUMDAR & al. (2008). They know that intraspecific quent than currently assumed. WANG & al. (2013) and morphs, either directly inherited or environment-induced, PURCELL & al. (2014) demonstrated for two ant subfami- have frequently been described as good species. Famous lies that alternate syndromes of gyne morphology and be-

42 havior, associated with either monogynous or polygynous tions to the geographic area where both phenotypes occur colony organization, are controlled by a single Y-like, non- sympatrically. Important variables to be considered in the recombinant chromosome that is not homologous in the analyses are within-nest sample size and the recognition investigated Solenopsis WESTWOOD , 1840 and Formica rate R. LINNAEUS , 1758 ants. Taxonomic data sets usually have an unequal within- Both monogenic and supergenic control are likely to nest sample size distribution. This depends on the hetero- have statistically similar effects on distribution of pheno- genous individual preferences of ant collectors and how types over the nests and populations when they follow a much material was available at all in historic and recent Mendelian inheritance. Within the taxonomic context, the collections. There may be, for example, in a certain obser- question of the underlying genetic mechanism is of lower vation sample ten nest samples with two workers, 15 sam- importance and simulating both ways by a simple Men- ples with three, 25 samples with four, one sample with delian model of monogenic dominant-recessive inheritance five, and eight samples with six workers. Direct calcula- appears an acceptable simplification. tion of Hardy-Weinberg frequencies appeared too difficult Five explanations for ant nests with mixtures of dis- for such irregular data sets. Accordingly, the prediction crete phenotypes: If a taxonomist collected material in was done in scenario (1) by a simulation that exactly re- nature and demonstrated the existence of discrete pheno- peated the unequal nest-sample size distribution in the ob- types forming clusters that can be identified with a mini- servation samples. mum error or are separated by wide gaps, he has to make The ratio of individuals with reliably determined pheno- credible that the clusters do not represent an intraspecific types within the total of the observation sample is called phenomenon. It was explained above that satisfying this re- the recognition rate R. A "reliably determined" phenotype quirement finds ideal conditions in eusocial insects with was assumed if the linear discriminant analysis of multiple perennial societies of defined kinship. Of particular interest morphometric data of an individual showed a posterior is here the abundance and distribution of phenotypically probability of p > 0.95 when testing a hypothesis previ- mixed nest populations. There are five possible scenarios ously formed by an exploratory data analysis. All nest for ant nests with a mixture of discrete phenotypes. The samples containing at least one reliably determined indivi- phenotypes may represent dual of the alternate phenotype were considered as mixed Scenario (1): genetically determined intraspecific morphs, and the remaining samples to contain only a single morph Scenario (2): intraspecific modifications induced by envi- irrespective if they contained individuals with p > 0.95 or ronmental factors, not. Scenario (3): the association of a temporary social para- A low recognition rate and a low mean within-nest site with a host species, sample size will reduce the frequency of identified mixed Scenario (4): the association of a permanent social para- nests and increase the number or supposed pure nests in site with a host species, and the observation sample. These changes have to be con- Scenario (5): a parabiotic association of two basically in- sidered in the simulation of the monogenic scenario (1) dependent (self-sustaining) species. and the predictions for the three scenarios of heterospe- I introduce here an analytical method named the DI- cificity (3), (4) and (5). MORPH test. The basic rationale of this test system to Scenario (1) – modeling "monogenic" intraspecific distinguish between heterospecificity and intraspecific di- dimorphism: I supposed above that a simple Mendelian morphism is comparing the observed nest-type frequen- model of monogenic dominant-recessive, pleiotropic in- cies (pure nests of phenotype 1 vs. mixed nests of pheno- heritance is a reasonable way to describe the distribution type 1 and 2 vs. pure nests of phenotype 2) with predic- of discrete character syndromes independent if there is tions for the five scenarios. These predictions are derived really such a mechanism underlying or if a linkage group from a mathematical model (scenario 1) and generaliza- of n non-recombinant structural genes has caused this ef- tions from empirical data (scenarios 2 to 5). The conside- fect. Firstly we consider which traits of ant biology are ration of nest type frequencies is restricted here to the important for the model. worker population because this is the only caste perma- (a) Ants are haplo-diploid, the males are hemizygous. nently available in sufficient sample sizes. In the follow- (b) Ant nests are perennial. Their worker populations ing I explain the biological background of the scenarios show comparably low annual fluctuations. (1) to (5) and present methods to predict the corresponding (c) Ant nests contain worker populations of closely nest-type frequencies. It will be shown below that predict- related individuals. Relatedness varies between two ex- ing scenarios (2) to (5) frequently lacks a precisely de- tremes: The population may represent the offspring of a fined basis but we should bear in mind that the intention single mother and a single father in monogynous-mono- of this paper is distinguishing heterospecificity from intra- androus nests but may have thousands of mothers and specific dimorphism. The analyses presented have the ex- fathers in polygynous-polyandrous nests. The social type plicit aim to avoid the production of taxonomic synonyms. of an ant nest affects the intranidal genotypic and pheno- Within the taxonomic context, it does not matter if the typic diversity. Polygynous nests may have more pheno- analyses confuse a parabiotic association with an associa- typical diversity than monogynous nests (SEIFERT 1991). tion of a temporary social parasite with a host. Considering that we aim here at taxonomic sampling in In the following I present the principles of the predic- larger geographic areas with several local populations, tion of nest-type frequencies. The individual worker mor- small-scale deviations should be statistically equalized in phologies are neutrally termed throughout the paper as a sufficiently large sample even in socially polymorphic "phenotypes" irrespective if they are finally identified as ant species. The problem is more thoroughly treated in morphs or species. The consideration restricts in all predic- Results and Discussion.

43 (d) Populations of monogynous ants with nuptial-flight dromes. EM may be mediated by most different environ- and strong dispersal capacity will approach panmixis (SEPPÄ mental factors: temperature, day length, water supply, quan- & PAMILO 1995, BOOMSMA & VAN DER HAVE 1998, VAN tity and quality of nutrition, pheromones and many others. DER HAVE & al. 2011). Yet, strong deviations from pan- Physical and chemical factors may have multiple effects. mictic behavior may occur in polygynous societies on a Sometimes an environmental factor may act analogous to small geographical scale due to population viscosity (LI- a transcription factor: Soy genistein in the diet of mice, AUTARD & KELLER 2001, CHAPUISAT & al. 1997). This is for instance, both changes the coat color and protects from caused by dominance of intranidal mating, weak long-range obesity (DOLINOY & al. 2006). dispersal of alates and differential queen acceptance ac- Assessment of EM is very difficult. It is not clear in cording to kinship value. Bearing in mind that our taxo- the first instance if a polymorphism is due to EM or direct nomic view includes samples from a larger geographical genetic inheritance. Mathematically predicting the frequen- area, highly polygynous ant species can be expected to cy of environmentally induced morphs in a metapopula- behave on the metapopulation level not too different from tion of ants or in a particular ant nest is a priori im- monogynous / monoandrous species. In other words, poly- possible. This would require intimate knowledge of the gyny should not affect the presented test system drama- environmental history of nest sites and on reasons, mech- tically which is supported by the following considera- anisms and parameters of the modifying process. Further- tions. The critical parameter in the test is the frequency more, rearing experiments under controlled conditions and of nests containing both phenotypes. High frequencies of parallel genetic investigations are needed. mixed nests indicate intraspecific dimorphism and low The perennial nature of ant nests, the long life expect- frequencies heterospecificity. Polygyny will increase the ancy of workers and the fact that they are reared in most chance that two phenotypes may occur within the same species from both spring and summer broods (or in the nest. Reduced exchange between viscose populations of tropics throughout the year) implies a high probability that highly polygynous ants, on the other hand, will violate preimaginal worker ontogenesis is subject to strongly vary- the panmixis condition and reduce the number of mixed ing environmental influences. Accordingly there should be nests. These opposing trends should cause some kind of quite many nests with phenotype mixtures. The problem compensation on the metapopulation level. caused by the impossibility of mathematically describing Which are the basic assumptions and procedures in the EM appears rather harmless if we consider which types of simulation of scenario (1)? The "monogenic" model as- possible errors occur and if these matter within the taxono- sumes panmictic behavior in the sympatric zone and dis- mic context. The central question is: May EM cause pheno- crete dimorphism. As ant males are hemizygous, a worker type distributions which are not separable from those in- offspring results from the following mating combinations: dicating heterospecificity? Confusion with the distribution AA × A, AA × a, Aa × A, Aa × a, aa × A, and aa × a. If p A of two separate species of self-sustaining species in which is the frequency of allele A and p a that of allele a, the mixed nests occur in less than 2% of all nests observed – 2 2 genotypes distribute according to p A + 2 p Apa + p a . The see point (5) below – is almost impossible. This type of er- simulation averages the data of two runs – the first run ror requires intraspecific separation in two very different assumed the rarer phenotype and the second run the more environments with very constant conditions throughout abundant phenotype to be homozygous recessive. Allele the season and EM to cause a very rigid switch from one frequencies were calculated alone from individual pheno- phenotype to the other without generating intermediate ex- type frequencies as they occurred in the observation sam- amples. There is not known any example for a very eury- ple in the sympatric zone. If there was in the analysis a potent ant species in Europe or worldwide with such a rigid total of 40 workers of phenotype 1 (genotype aa ), and intraspecific habitat segregation. The same arguments ex- 120 workers of phenotype 2 (genotypes Aa and AA ), the clude a confusion with the distribution for temporary so- frequency of allele a was the square root of (40 / 160) = cial parasitism in which mixed nest form less than 1% of the 0.5. Genotypes of parents and the resulting worker offspring total – see background information for scenario (3) given were simulated by random number generation. Then, using below. Confusion with permanent social parasites requires the recognition rate R, it was determined at random if the EM to generate many pure nests of one phenotype 1 ("host"), applied morphological classification system could posi- fewer or much fewer nests with phenotype mixtures ("host" tively identify a genetically determined phenotype. If there + "parasite") but never to generate pure nests with pheno- was, for instance, a genotypic prediction of 100 workers type 2 ("parasite"). Such a scenario is highly unlikely for of phenotype 1 in the total data set and the recognition conventional EM. Yet, infestion with endoparasites may rate R was 90%, only 90 workers in the simulation were generate similar distributions. These cases, at least those identified as phenotype 1 with the individual decisions de- which are sufficiently studied today (e.g., infestions by Mi- termined by random numbers. As a consequence from re- crosporidium or Mermis , see CSŐSZ & MAJOROS 2009), cognition rates below 1.0 and the low number of workers are not really dangerous for experienced taxonomists. Good analysed within the nest samples, the frequency of pre- taxonomists usually recognize different types of pathoge- dicted mixed nests will be reduced and the number of pure nic phenotype deformations. Furthermore, "uncontrolled" nests increased in deviation from the ideal Hardy-Weinberg variability or intermediacy of pathogenic phenotypes may situation. The prediction of scenario (1) was calculated as additionally signalize an intraspecific phenomenon. mean of 1000 repeats and compared with the really ob- The remaining, more likely, type of error, confusing served situation. EM with direct genetic inheritance, is regrettable from the Scenario (2) – intraspecific dimorphism induced by perspective of understanding ant biology but does not mat- environmental factors: Environmental modification (EM) ter from a taxonomic point of view. Apart from using the may occasionally cause morphs with distinct character syn- prediction formulae presented below, I recommend to fol-

44 low a simple rule of thumb: If there are more than 4% of ginal habitats or such with strong anthropogenic impact all nest samples in a larger geographic area mixed with two where hosts but no parasites are present. Considering that discrete phenotypes and if there are no indications for a average myrmecologists avoid collecting in such poor hab- social parasite to be involved, an ant taxonomist should re- itats with low species diversity and that ant taxonomists frain in the first place from considering them as different evaluate just these collections, it seems adequate to correct species and should investigate what is behind the pheno- in the prediction the parasite-host ratio to 1 : 40 (0.025). menon. Temporary social parasitism in the genus Formica LIN - Scenarios (3) and (4) – association of a temporary NAEUS , 1758 differs in some aspects from the situation in or permanent social parasite species with a host: Intra- Lasius . In the Palaearctic zone, temporary social parasites nidal co-occurrence of ants with distinct phenotypes is also parasitizing ants of the subgenus Serviformica FOREL , 1913, given in nests mixed of temporary or permanent social belong to the subgenera Coptoformica FOREL , 1913, Rapti- parasites and their host species. Morphologies of these so- formica FOREL , 1913 and Formica s.str. (i.e., the red mound- cial parasites differ from their hosts in a way that hetero- building wood ants). The latter three subgenera colonize specificity is no question. Hence, there should normally not a new site by temporary social parasitism in Serviformica , exist a taxonomic problem on the host vs. parasite level. may then propagate by colony fission and will subsequently Here, we ask for the hypothetical case of social parasitism reduce the populations of the subordinate Serviformica spec- where host and parasite are very similar and where no ies by aggressive interference, competition, predation or ac- special morphological characters have been developed in- tive raiding. The extremes of such local cycles may vary dicating a parasite identity. Such a situation will occur in between no social parasites present on a site and almost initial stage of parasite evolution and should be generally complete displacement of host species. As particular local rare. There is no question that such cases are cleared up situations are not relevant for our taxonomic considera- in the most conclusive way by ethological and genetic in- tions, we have to ask what are typical or average figures vestigations. However, as a taxonomist frequently has only for a larger geographic area. Within the system of 232 series of dead and very old museum material at hand but test plots in Central Europe, the collective density of all no deeper biological information, the distribution of pure species of Formica s.str., Raptiformica and Coptoformica and mixed nest samples may provide a hypothesis on a was 0.231 nests / 100 m² while the collective density of all possible biological background. species of Serviformica was 4.67 / 100 m². This means a Scenario (3) – predicting the distribution of tempor- ratio of temporary parasites versus hosts in the genus For- ary social parasites and host: In case of species always mica of about 1 : 20, precisely 0.0495. This is twice the founding their nests by temporary social parasitism and value of 0.025 assumed for Lasius . Accordingly we may considering a larger geographic range, mixed host-parasite fix as generalization for both genera a value 0.0372. An nests are much rarer then pure parasite nests and the latter obvious figure for the relation of mixed host + parasite themselves are much rarer than pure host nests. The situa- nests vs. pure parasite nests cannot be given for temporary tion in temporary social parasites belonging to the subgen- social parasitism in Formica . In the subgenera Formica era Chthonolasius RUZSKY , 1913 and Austrolasius FABER , s.str. and Coptoformica this ratio is much lower than the 1967 which found colonies in hosts of the subgenera La- Lasius figure of 0.0416 because of frequent nest founda- sius RUZSKY , 1913 and Cautolasius WILSON , 1955 pro- tion by colony-fission and in the subgenus Raptiformica vides an exemplary picture. A survey of collection ma- much higher because of facultative slave raids. For a gen- terial and data files present in SMN Görlitz discovered eralization we can only use the figure of Lasius . only 21 nest samples mixed of social parasites and host For testing if the nest-type frequencies could indicate but 505 pure samples of the social parasites when all fif- temporary social parasitism, a prediction was built up teen parasite and nine host species are considered collec- with the following assumptions. The observed number of tively. This is a ratio of 1 : 24 or 0.0416. The general rar- pure nests of the rarer phenotype determines the assumed ity of mixed parasite-host nests is explained by the fact number of pure parasite nests P. According to the data de- that the parasite needs the host only for the initial stage of scribed above, the assumed number M of mixed parasite- colony foundation. After the host queen has been killed by host nests should then be M = 0.0416*P and the number the parasite or her own children, the Lasius parasite queen of pure host nests H should be H = P*26.9. The precise consumes the host eggs. When a sufficient population of algorithm of calculating the prediction for the scenario of temporary parasite workers has been reared by the host temporary social parasitism is presented in Table 1. The workers, the parasite workers begin to kill the remaining data are finally standardized in a way that the sum of host workers. Consequently, the transition to a pure para- samples in the prediction and observation are equal. site colony is frequently completed in Lasius as soon as The empiric data in Lasius and Formica described 12 - 14 months after colony foundation (CRAWLEY 1909, above were collected under nearly ideal conditions: Host HÖLLDOBLER 1953; B. Seifert, unpubl.). The relation be- and parasite could be identified with recognition rates of tween established nests of the temporary parasite and host 1.0 and the within-nest sample size of these field collec- nests can be derived from nest density data from 232 study tions was much higher than the within-nest sample size plots over all habitat types in Central Europe (B. Seifert, of NUMOBAT-evaluated workers. As it was done in the unpubl.). Over all Lasius species, the mean collective dens- simulation for monogenic dimorphism, we have to con- ity of Chthonolasius and Austrolasius social parasites was sider in the predictions for scenarios (3), (4) and (5) that 0.32 nests / 100 m² and 27.3 nests / 100 m² in the host a low mean sample size and recognition rates below 1 will species. This is a relation of 1 : 86 or 0.012. It must be reduce the frequency of recognized mixed nests which are noted that this figure was calculated from a survey aiming defined as pure if a mixed status cannot be shown on the at ecological data, containing a significant fraction of mar- p > 0.95 level. Conversely, the frequency of pure nests of

45 Tab. 1: Variables and algorithms to calculate predictions for nest-type frequencies of three sce- narios of heterospecific intranidal associations in ants.

Observation sample: P1 = frequency of pure nests of the rarer phenotype 1 M = frequency of mixed nests with both phenotype 1 and 2 P2 = frequency of pure nests of the more abundant phenotype 2 T = total number of samples ‰ T = P1 + M + P2 Correction for influence of l ow recognition rates and low and unequal within -nest sample size:

MRE = number of mixed nests predicted in the simulation of scenario (1) operating with the really found recognition rate and really observed nest sample size distribution

MID = number of mixed nests predicted in the simulation of scenario (1) assuming "ideal" conditions: a recognition rate of 1.0 and a sample size of always 10 workers per nest.

C1 = correction factor describing the reduction of mixed nest frequency ‰ C1 = M RE / M ID Algorithm for prediction of frequencies in the parabiosis scenario:

M1 = interim value of mixed (parabiotic) nests ‰ M1 = 0.01 * T

P1 1 = interim value of pure nests of phenotype 1 ‰ P1 1 = P1 / (P1 + P2) * 0.99 * T

P2 1 = interim value of pure nests of phenotype 2 ‰ P2 1 = P2 / (P1 + P2) * 0.99 * T

M' = corrected (final) value of mixed nests ‰ M' = M 1 * C 1

D = difference caused by correction ‰ D = M 1 – M'

P1 ' = final value of pure phenotype 1 nests ‰ P1 ' = P1 1 / (P1 1 + P2 1) * D + P1 1

P2 ' = final value of pure phenotype 2 nests ‰ P2 ' = P2 1 / (P1 1 + P2 1) * D + P2 1 Algorithm for prediction of frequencies in the temporary social parasitism scenario: st P1 = 1 interim value of pure parasite nests ‰ P1 = P1 st M1 = 1 interim value of mixed parasite -host nests ‰ M1 = 0.0416 * P1 st H1 = 1 interim value of pure host nests ‰ H1 = P1 * 26.9

C2 = correction factor adjusting P 1 + M 1 + H 1 to T ‰ C2 = T / (P 1 + M 1 + H 1) nd P2 = 2 interim value of pure parasite nests ‰ P2 = C 2 * P 1 nd M2 = 2 interim value of mixed parasite -host nests ‰ M2 = C 2 * M 1 nd H2 = 2 interim value of pure host nests ‰ H2 = C 2 * H 1

M'' = final value of mixed parasite -host nests ‰ M'' = M 2 * C 1

D = difference caused b y correction ‰ D = M 2 - M''

P1 '' = final value of pure parasite nests ‰ P1 '' = P 2 / (P 2 + H 2) * D + P 2

P2 '' = final value of pure host nests ‰ P2 '' = H 2 / (P 2 + H 2) * D + H 2 Algorithm for prediction of frequencies in the permanent social par asitism scenario: P1 ''' = final value of pure parasite nests ‰ P1 ''' = 0 st H1 = 1 interim value for pure host nests ‰ H1 = P2 st M1 = 1 interim value for mixed parasite -host nests ‰ M1 = 0.02 *P2

C2 = correction factor adjusting P1 ''' +M 1+H 1 to T ‰ C2 = T / (M 1 + H 1) nd M2 = 2 interim value for mixed parasite -host nests ‰ M2 = C 2 * M 1 nd H2 = 2 interim value for pure host nests ‰ H2 = C 2 * H 1

M''' = final value of mixed parasite -host nests ‰ M''' = M 2 * C 1

D = differe nce caused by correction ‰ D = M 2 - M'''

P2 ''' = final value of pure host nests ‰ P1 ''' = H 2 + D the alternate phenotypes will increase. In order to estimate samples containing 10 workers. The frequencies obtained the influence of lower recognition rates and lower within- in these runs were then compared with the frequencies from nest sample size of a particular case, the simulation of the runs under real conditions with recognition rates ranging dimorphism scenario (1) was repeated assuming near to between 0.820 and 0.993 and a mean sample size between ideal conditions: A recognition rate of 1.0 and all nest 2.44 and 10.0 (see Tab. 2). These simulations showed clear

46 differences between the ten cases reported here. The strong- observation sample determines the number of pure host est reduction of the number of identified mixed nests, nests H in the prediction. The number of mixed parasite- down to 80.8% of the ideal value, occurred in the Phei- host nests M is then M = 0.02*H and the number of pure dole case under a recognition rate of 0.829 and a mean parasite nests is P = 0. The sum of nest-type frequencies sample size of 2.80. The weakest reduction, down to 99.3% in the prediction is equalized to the sum of nest-type fre- of the ideal value, occurred in the Formica lugubris case quencies in the observation sample and finally corrected under a recognition rate of 0.914 and a mean sample size to for frequency distortions caused by recognition rates < 1 of 6.21. These reduction factors were used to finally cor- and small within-nest sample size. rect the nest-type frequencies in all three predictions for Scenario (5) – parabiotic associations of two inde- heterospecificity. Details of the algorithms are presented pendent species: Intranidal co-occurrence of ants with dis- in Table 1. However, these corrections had only a minute tinct phenotypes may also represent a parabiosis. Parabiotic effect on frequencies and test parameters – mainly because species share nest space, foraging trails and food sources the frequency of mixed nests in the three heterospecifici- but keep their brood chambers separate. It is important to ty scenarios was already very small before the correction. note that all long-lived parabiotic associations known so Scenario (4) – predicting the distribution of perma- far in ants involve very distantly related, immediately se- nent social parasites and hosts: Permanent social para- parable species, belonging to different subfamilies. Most sitism includes dulotic species and inquilines. All obliga- frequent are parabioses between Crematogaster LUND , 1831 tory dulotic species known so far differ morphologically (subfamily Myrmicinae) and Camponotus (subfamily For- from the host species in a way that a taxonomist can safely micinae). At least seven species of Crematogaster and six conclude on heterospecificity. Genus-level differences are species of Camponotus are reported worldwide to live in the rule. Yet, some species of less evolved inquilines may a stable parabiosis (KAUDEWITZ 1955, BERNARD 1968, be rather similar to the host species. Important for the pre- SWAIN 1980, SEIFERT 2007, VANTAUX & al. 2007, MEN - diction is that a permanent social parasite cannot live alone ZEL & al. 2008, MENZEL & al. 2014; F. Menzel, unpubl.). and pure nests of the rarer phenotype will not occur. As a In northern Italy and the Balkans, mixed nests of three Cre- consequence, only mixed host-parasite and pure host nests matogaster species with Camponotus lateralis made up 12% are possible with the latter being much more abundant over of 113 nests found (data and collection material provided larger territories. These facts alone exclude a confusion by courtesy of H.C. Wagner & A. Vesnic). Other exam- with temporary social parasitism and parabiosis. Yet, we ples of stable parabioses are those of Crematogaster with have to consider and describe this scenario because nest- Dolichoderus LUND , 1831 (SWAIN 1980) and of Pseudo- type frequencies of monogenic dimorphism may be inse- myrmex LUND , 1831 with Camponotus (GALLEGO -ROPERO parable from those of permanent social parasitism when & FEITOSA 2014). Furthermore, the inquiline relations of the allele frequency of the rarer morph in the dimorphism Polyrhachis SMITH , 1857 with Diacamma MAYR , 1862 scenario is about 0.1. (MASCHWITZ & al. 2000) and of Polyrhachis with Rhyti- Data of four parasite species are available. These in- diponera MAYR , 1862 (MASCHWITZ & al. 2003) most dicate strong variation of the ratio of mixed parasite-host probably evolved from parabiotic associations. against pure host nests depending upon geographical scale The fact that all long-term stable parabioses known and population dynamics. The parasitation ratio of Tetra- worldwide are between members of different subfamilies morium nests by the inquiline Strongylognathus testaceus is intriguing and a short excursus on the possible reasons is about 4% in some parts of the German countries Bran- should be allowed here. Contemporary evolution theories denburg and Sachsen (SEIFERT 2007) whereas it was only assume reinforcement to be a significant segregating fac- 0.33% in 900 checked Tetramorium nests in southern tor during speciation (DOBZHANSKY 1940, SERVEDIO & France (BERNARD 1968). The inquiline Myrmica hirsuta NOOR 2003, COYNE & ORR 2004). Reinforcement occurs may locally parasitize 40 - 50% of Myrmica sabuleti nests when natural selection strengthens behavioral discrimina- (SEIFERT 1988, MÜLLER 2003) but the Central European tion to prevent costly interspecies matings, such as when average over all M. sabuleti populations is surely not above matings produce sterile hybrids. For instance, reinforce- 2% (SEIFERT 2007). The ratio of nests of the dulotic spe- ment in Drosophila for female mating discrimination is cies Harpagoxenus sublaevis (NYLANDER , 1849) against enhanced by natural selection. Accordingly, it seems plaus- the Leptothorax MAYR , 1855 host nests was 1.5% against ible to assume that selection should act to avoid too inti- 98.5% in 232 test plots in Central Europe but at local spots mate contacts of reproductive cycles between closely re- Harpagoxenus grew to 5% (B. Seifert, unpubl.). The re- lated ant species. Development of parabiotic associations lation of nests of the dulotic species Polyergus rufescens is more likely when there is no longer any danger for in- (LATREILLE , 1798) against Serviformica host nests was terspecific hybridization as it is given between members 1.5% against 98.5% (accidentally the same figure as in of different subfamilies. Another, may be the more pow- Harpagoxenus ) in 16 study plots in limestone grass- erful, component of explanation should be an ecological lands of Thüringen and Sachsen-Anhalt but locally Poly- one: Overlap of ecological niches and interspecific aggres- ergus achieved 3.3% (B. Seifert, unpubl.). From these data sion grow with increasing relatedness. This leads to mu- and referring to a larger geographical scale it seems a rea- tual spacial exclusion or active displacement of closely sonable generalization to assume 2% of mixed parasite- related species (for ants shown by SEIFERT 1987). host nests and 98% of pure host nests. Mixed nest populations of closely related ant species The precise algorithm of calculating the prediction for that are self-sustaining in mature colonies are exceptional the scenario of permanent social parasitism is presented and short-lived. They have been observed, for example, in in Table 1. The algorithm firstly assumes that the number several Formica rufa group ants (HAGEMANN & SCHMIDT of pure nest samples of the more abundant morph in the 1985, SEIFERT 1991, CZECHOWSKI 1996), Temnothorax

47 MAYR , 1861 (PUSCH & al. 2006), Messor FOREL , 1890 with posterior probabilities of p > 0.95 were considered as (STEINER & al. 2011) or in Myrmica LATREILLE , 1804 "reliably classified". (BAGHERIAN YAZDI & al. 2012). The majority of such Phenotyping included all material from both the sym- parabiotic cases represent a transitionary stage of a nest patric and allopatric ranges, but the prediction scenarios, shifting from one species population to another after colony including calculation of allele frequencies, considered only usurpation by a heterospecific queen, peaceful adoption of material from geographic areas for which sympatric oc- a queen by an orphaned nest or a stage after allopleome- currence of the alternate phenotypes was directly confirmed trotic colony foundation. Raiding of heterospecific brood or appeared probable according to the large scale distribu- could also explain temporarily mixed societies as it was tion pattern. Phenotype frequencies and the resulting allele shown for Pogonomyrmex MAYR , 1868 and Messor (HÖLL - frequencies are based not only on reliably determined in- DOBLER & MARKL 1990; RISSING & POLLOCK 1991). The dividuals but on any individual with posterior probability frequency of parabiotic associations of closely related spe- of p > 0.5. LDA, PCA and k-means clustering were run cies is generally < 2% of all nests observed for both in- with the software package SPSS 15.0 and NC-Ward and volved species – here I fix 1% in the prediction algorithm. NC-K-means in the software package R freely available Possible genetic and ecological reasons leading to this small under the GNU / GPL license from http://sourceforge. figure were explained above. net/projects/agnesclustering/. Removal of allometric vari- For our taxonomic purpose, we need only to consider ance (RAV) was performed in strongly allometric species the temporary parabioses of closely related species. If this according to the method of SEIFERT (2008). type of association is confirmed by phenotype distribu- Observed and predicted distributions of nest types were tions, an argument for heterospecificity is given. It is not compared by the X² test of independence according to SO- relevant in this context if 1% of interspecifically mixed KAL & ROHLF (1995). There is a problem in X² statistics nests represent true parabioses or a determination error with very small samples relevant for the data sets used in and it does also not matter within the taxonomic context this paper – frequently the number of mixed nests in the if a parabiosis is confused with temporary social parasitism. observation data set and in the predictions for heterospe- The algorithm predicting nest-type frequencies for tem- cificity is below five in a certain cell (see Tab. 2). Small porary parabioses of closely related species is presented frequencies are supposed to result in disproportionately in Table 1 and includes an equalization of the total number increased X² values and thus in a false rejection of the null of samples in the observation and prediction and a final hypothesis. Another unsolved problem occurs with possi- correction for frequency distortions caused by recognition ble differences in the degrees of freedom (df) because the rates < 1 and small within-nest sample size. three predictions for heterospecificity were generated with data from the observation sample. This should result in a Material and methods reduction of df. The 3 × 2 table used in all the tests ba- Analysis methods: In a first analytical step phenotyping sically has df = 2. The observation sample has three sin- was done in material both from sympatric and allopatric gular parameters P1, M, P2 and one collective parameter ranges. There were two alternative ways, a direct and an T = P1 + M + P2. The prediction (simulation) for gene- indirect one, to demonstrate discrete phenotypes. The di- tically mediated dimorphism does not use any data of the rect approach started on the level of worker individuals observation sample. Accordingly, the basic figure of df = 2 and formed initial hypotheses on discrete phenotypes by should remain unchanged. The prediction for parabiosis the exploratory data analyses principal component analysis used P1 and P2, the prediction for temporary social para- (PCA) and k-means clustering. The hypotheses provided sitism P1 and T and that for permanent social parasitism by exploratory data analyses were then tested by a single P2 and T. There is no rationale or algorithm available tel- run of a linear discriminant analysis (LDA). All pheno- ling us in which way df has to be reduced in the hetero- types with posterior probabilities of p > 0.95 were con- specificity scenarios and we have to leave the question sidered as "reliably recognized" and their proportion open. However, the strong differences in the basic struc- from the total is the recognition rate R. This direct way ture of the predictions should provide a powerful differ- of analysis was applied when phenotypes differed more entiation of the scenarios and probably make the df ques- obviously. tion less important. Alternatively, in more hidden cases of polymorphism These two problems can be circumvented if we follow such as in Cardiocondyla elegans , when starting the ana- alternative approaches (e.g., AKAIKE 1973, BURNHAM & lysis on the individual level provided no clear cluster se- ANDERSON 2002) which aim to estimate the quality of a paration, the analysis began by running the exploratory data model, relative to each of the other proposed models with- analyses NC-Ward clustering and NC-K-means clustering out assessing the quality of a model in an absolute sense. (SEIFERT & al. 2014b) which operate on the nest-sample Here I select which of a range of possible predictions best level. Clusters resolved in this way on the nest-sample lev- fits the observation data rather than testing whether obser- el are likely to be correlated with phenotypes on the indivi- vations differ from predictions of a particular scenario more dual level even if a considerable fraction of nest samples than expected by chance. This solution is allowed by the was a mixture of phenotypes. These NC clusters were ac- following observation. If sample size is multiplied in all cepted as hypothesis on individual phenotypes and tested three nest-type frequencies by a factor that the smallest in a first LDA in all worker individuals. If there are mixed sample becomes a value of 10 or 100, there is a full cor- nests in the material, this initial LDA will give a first im- relation of the X² values with those of the primary data pression of the situation. Accepting the classifications of set. In other words, the ranking of X² values is indepen- the first LDA as hypothesis, a second LDA was run and dent from absolute sample size. This ranking has a linear the analysis terminated. As in the direct way, all phenotypes correspondence with the Akaike Information Criterion as

48 long as we use the same total number of nests in all four curs alone, the predictions were restricted to 206 nest sam- scenarios. Accordingly, the smallest X² values always in- ples with 772 workers from the supposed sympatric range dicate the most likely of the four scenarios. in Europe – in detail 343 workers of phenotype 1 and 429 Four cases of intraspecific dimorphism are presented workers of phenotype 2. Absolute size, two shape and here – three of these have produced (or are likely to pro- three setae characters were numerically recorded (see Ap- duce) erroneous descriptions of species while the fourth pendix S1). case is more hidden. I also present four cases of taxo- Case 6: the cryptic species Temnothorax crassispinus nomically recognized pairs of cryptic species and one ex- (KARAVAJEV , 1926) and T. crasecundus SEIFERT & ample of more easily separable related species is added to CSŐSZ , 2015: The primary analysis included 203 nest sam- show the difficulty to distinguish between parabiosis and ples with 603 workers from the whole Westpalaearctic range temporary social parasitism. of both phenotypes (SEIFERT & CSŐSZ 2015). The predic- Case 1: intraspecific dimorphism in Camponotus tions were restricted to 84 nest samples from the sympa- lateralis (OLIVIER , 1792): The primary analysis included tric range in Europe (Greece, Bulgaria, Romania, and Mol- 64 nest samples containing 86 workers of phenotype 1 and dova) containing 93 workers of phenotype 1 (= Temno- 64 workers of phenotype 2 from Spain, southern France, thorax crassispinus ) and 159 workers of phenotype 2 (= T. Italy, Croatia, Montenegro, Macedonia, Bulgaria, Greece, crasecundus ). Absolute size and 29 shape characters were Cyprus, Asia Minor and Georgia. Thirteen morphometric numerically recorded (for details see SEIFERT & CSŐSZ characters including shape, microsculpture and pilosity 2015). characters were numerically recorded (see supplementary Case 7: the cryptic species Temnothorax luteus (FO- file Appendix S1, available as digital supplementary mate- REL , 1874) and T. racovitzai (BONDROIT , 1918): The pri- rial to this article, at the journal's web pages). mary analysis included 64 nest samples from the whole Removal of allometric variance was performed by di- European range with 178 workers of both phenotypes (SEI - phasic functions because there is a major-minor dimorphism FERT & al. 2014a). The predictions were restricted to 53 in this species superimposing the shape-pilosity dimorph- nest samples from the sympatric range in Spain and France ism. The observation sample to be compared with the pre- containing 73 workers of phenotype 1 (= Temnothorax dictions considered 53 samples from sympatric range. racovitzai ) and 80 workers of phenotype 2 (= T. luteus ). Case 2: intraspecific dimorphism in Lasius umbra- Absolute size and 17 shape characters were numerically re- tus (NYLANDER , 1846): The primary analysis included 113 corded (for details, see SEIFERT & al. 2014a). nest samples from Europe with 57 workers of phenotype 1 Case 8: two cryptic species of the Pheidole pallidula and 289 of phenotype 2. The observation sample to be (NYLANDER , 1849) complex: The primary analysis included compared with the predictions was reduced to 104 sam- 72 nest samples from the whole Palaearctic range of the ples from the assumed sympatric range of both phenotypes two species from Portugal to Middle Asia with a total of that included Austria, Czechia, Great Britain, Germany, 191 major workers analysed. The predictions were restricted Poland, Slovakia, Slovenia, Sweden, and Switzerland. Four- to 43 nest samples from the sympatric range in the Bal- teen morphometric characters including shape, pilosity and kans and Asia Minor containing 58 workers of phenotype 1 pubescence characters were numerically recorded (see Ap- (= P. sp. BALC) and 65 workers of phenotype 2 (= P. sp. pendix S1). KOSH). Absolute size, 15 shape characters and one seta Case 3: intraspecific dimorphism in Formica lugu- character were numerically recorded (see Appendix S1). bris ZETTERSTEDT , 1838: The primary analysis included Case 9: the related species Myrmica scabrinodis NY- 77 nest samples from Fennoscandia with 108 workers of LANDER , 1846 and M. vandeli (BONDROIT , 1920): Myr- phenotype 1 and 369 workers of phenotype 2. Since pheno- mica scabrinodis and M. vandeli are similar but no cryptic type 1 was not found in Norway, the predictions were re- species. Experienced researchers can separate the two spe- stricted to Sweden and Finland with a total of 67 nest sam- cies with a low error rate by simple eye inspection using ples. Seven morphological characters including four shape a stereomicroscope. I used a data set of Wolfgang Münch and six pilosity characters were numerically recorded (see who inspected about ten workers per nest in 3763 nest Appendix S1). samples of both species collected in Baden-Württemberg / Case 4: intraspecific dimorphism in Cardiocondyla Germany. This area is in the centre of the sympatric range. elegans EMERY , 1869: The analysis included 48 nest sam- The case is presented to show the difficulty to separate ples with 56 workers of phenotype 1 and 63 workers of between the predictions for parabiosis and temporary so- phenotype 2 from Iberia, France, and Italy. This is the cial parasitism. whole known range of Cardiocondyla elegans . Results and discussion Fourteen morphological characters – head size, eleven shape and two pubescence characters –were numerically Table 2 shows the results of the DIMORPH test for all nine recorded (see Appendix S1). cases treated here. Remember that the X² test statistic in Case 5: the cryptic species Formica pressilabris NY- these 3 × 2 tables correctly indicates the ranking of simi- LANDER , 1846 vs. F. foreli BONDROIT , 1918: The pri- larities when the total number of samples is equal in each mary analysis included 252 nest samples from the whole case. Lowest X² values indicate which prediction among Palaearctic range with 955 workers – 481 workers of phe- the four presented is most similar to the observation. The notype 1 (= F. pressilabris ) and 476 workers of phenotype DIMORPH test indicated intraspecific dimorphism in the 2 (= F. foreli ). Excluding samples from north temperate first four cases and heterospecificity in the last five cases. regions of Fennoscandia and Siberia where phenotype 1 Note that the presented parabiosis scenario refers to intra- occurs alone and samples from the Apennine, Asia Minor nidal associations of closely related species in which higher and the Central Asian Mountains where phenotype 2 oc- frequencies of mixed nests have not been observed so far.

49 Tab. 2: Data of the DIMORPH test. Comparison of observed nest-type frequencies with predicted frequencies for parabi- osis of closely related species, temporary social parasitism, permanent social parasitism and intraspecific dimorphism. P1 = number of nests with only phenotype 1, M = number of nests with phenotype mixtures, P2 = number of nests with only phenotype 2, R= recognition rate on the p > 0.95 level, m = mean within-nest sample size. X² is the test statistic of a Chi- squared test of independence according to SOKAL & ROHLF (1995). X² data correctly show the ranking of similarity be- tween observation and prediction with lowest values (printed in heavy type) indicating which prediction is the most probable.

Case Observed Prediction heterospecificity Prediction dimorphism Parabiosis Temporary Permanent social parasite social parasite Camponotus lateralis , P1 20 23.32 1.90 0 15.41 morph 2 (P1) vs. morph 1 (P2) M 8 0.52 0.08 1.03 15.99 P2 25 29.16 51.02 51.97 21.59 R, m 0.993, 2.68 X², p 7.14, 0.028 31.63, 0.001 34.83, 0.001 3.51 , 0.170 Lasius umbratus , P1 9 10.91 3.72 0 8.76 Compacta morph (P1) vs. M 19 0.97 0.14 1.90 17.19 normal morph (P2) P2 76 92.12 100.13 102.10 78.04 R, m 0.974, 3.01 X², p 18.01, 0.001 24.08, 0.001 26.82, 0.001 0.05 , 0.744 Formica lugubris , P1 15 17.46 2.40 0 9.34 Hippie morph (P1) vs. M 10 0.67 0.10 1.30 15.24 normal morph (P2) P2 42 48.87 64.50 65.70 42.42 R, m 0.914, 6.21 X², p 8.86, 0.012 23.58, 0.001 26.89, 0.001 2.41 , 0.283 Cardiocondyla elegans , P1 19 21.03 1.57 0 16.92 slender morph (P1) vs. M 5 0.41 0.06 0.80 11.85 thick morph (P2) P2 24 26.56 42.37 47.20 19.23 R, m 0.916, 2.44 X², p 4.13, 0.126 24.55, 0.001 29 .60, 0.001 3.43 , 0.177 Formica pressilabris P1 85 87.22 7.36 0 65.26 (P1) vs. F. foreli (P2) M 7 1.80 0.27 3.54 53.35 P2 114 116.98 198.36 202.46 87.39 R, m 0.868, 3.76 X², p 3.14 , 0.202 94.28, 0.001 110.90, 0.001 41.71, 0.001 Temnothorax crassispinus P1 30 30.47 3.01 0 21.83 (P1) vs. T. crasecundus (P2) M 2 0.70 0.11 1.37 19.35 P2 52 52.83 80.88 82.63 42.82 R, m 0.849, 3.05 X², p 0.64 , 0.605 30.03, 0.001 37.09, 0.001 16.28, 0.001 Temnothorax luteus (P1) P1 26 26.24 1.90 0 18.19 vs. T. ravovitzai (P2) M 1 0.52 0.08 1.06 14.80 P2 26 26.24 51.02 51.94 20.01 R, m 0.938, 2.98 X², p 0.15 , 0.734 29.73, 0.001 34.64, 0.001 14.21, 0.001 Pheidole sp. BALC (P1) P1 21 21.31 1.57 0 16.60 vs. Pheidole sp. KOSH (P2) M 1 0.36 0.06 0.70 10.11 P2 22 22.32 42.37 43.29 18.28 R, m 0.829, 2.80 X², p 0.31 , 0.691 23.21, 0.001 28.00, 0.001 8.32, 0.016 Myrmica vandeli (P1) P1 225 225.02 134.67 0 93.38 vs. M. scabrinodis (P2) M 38 37.63 5.60 73.78 292.61 P2 3500 3500.35 3622.73 3689.22 3377.02 R, m 0.900, 10.0 X², p 0.00 , 0.779 48.88, 0.001 241.43, 0.001 252.69, 0.001

More detailed comments on each case are given in the dividuals by 100% with a recognition rate of 100% for pos- following. terior probabilities of p > 0.95. The discriminant values were separated by a wide gap, being -2.712 ± 0.704 [-5.26, Case 1: Intraspecific shape-setae-pubescence -0.85] in 86 workers of phenotype 1 and 3.644 ± 1.196 dimorphism in Camponotus lateralis [0.77, 6.14] in 64 workers of phenotype 2. Phenotype 1 is The separation of the phenotypes in a PCA was complete a fixed combination of missing or reduced scape pilosity, (Fig. 3) and the LDA confirmed this clustering in 150 in- of a strongly developed scape base extension, a more elon-

50

Fig. 1: Intraspecific dimorphism in Cam- ponotus lateralis , head of two major work- ers shown. Left: Phenotype 1 with re- duced pilosity of scape and posterior head, a strongly developed scape base extension and more sinusoidally curved frontal cari- nae compared to phenotype 2 (right). Pho- tos from antweb.org, taken by Michele Esposito. Right: CASENT0914262, spe- cimen misidentified by L. Borowiec as Camponotus honaziensis . Left: CASENT- 0914267.

Fig. 2: Intraspecific dimorphism in Cam- ponotus lateralis , mesosoma of two ma- jor workers shown. Upper: Phenotype 1 with a longer, weakly convex dorsal plane of propodeum, shorter pubescence and more developed pilosity compared to phe- notype 2 (lower). Photos from antweb. org, taken by Michele Esposito. Lower: CASENT0914262, specimen misidenti- fied by L. Borowiec as Camponotus ho- naziensis . Upper: CASENT0914267.

gated scape, a longer, weakly convex dorsal plane of pro- ralis phenotype 2. Table 3 shows a number of striking dif- podeum, shorter pubescence, sinosoidally formed frontal ferences in RAV-corrected shape variables. carinae and a broader head (Figs. 1, 2). Phenotype 2 com- A proportion of 15% mixed nests for an average within- bines profuse scape pilosity with a weakly developed scape nest sample size of only 2.68 is a very high figure. Intra- base extension, a shorter scape, a shorter, strongly con- specific monogenic dimorphism is by far the most prob- vex dorsal plane of propodeum, much longer pubescence, able scenario for phenotypes 1 and 2 indicated by much more linear frontal carinae and a broader head (Figs. 1, 2). lower X2 values compared to the three scenarios for hetero- Both phenotypes provide the appearance of closely related specificity (Tab. 2). In this context it should be mentioned but clearly separable species and they were misinterpreted that nest samples from Bulgaria, where only phenotype 1 as such in the past. Having at hand two clean samples of was observed, were included into the sympatric range. An phenotype 2, SEIFERT (2007) hypothesized phenotype 2 exclusion of this area would increase the percentage of to represent a species " Camponotus lateralis sp. 2". Re- mixed samples in the observation sample and would make cently, BOROWIEC & SALATA (2014) misidentified pheno- the fitting to the dimorphism scenario even stronger and type 2 as C. honaziensis KARAMAN & AKTAÇ , 2013 (see not change the rejection of the other models. There is no CASENT0914262 in www.antweb.org, and Figs. 1 and 2). clear approach to exclude environmental modification of Investigation of three workers from the holotype nest of having generated this dimorphism but the absence of in- C. honaziensis from Honaz Dagi National Park, 37.467° termediate morphs strongly suggests a direct genetic mech- N, 29.217° E, 1195 m, leg. Karaman 2007.07.15, No 07 / anism. Yet, from a taxonomical perspective the true reason 2344 clearly confirmed the heterospecificity from C. late- for the development of this dimorphism does not matter.

51

Fig. 4: Principal component analysis of phenotype 1 (Com-

pacta morph, black discs, n = 57) and phenotype 2 (normal Fig. 3: Principal component analysis of phenotype 1 (white morph, grey squares, n = 289) of Lasius umbratus con- squares, n = 86) and phenotype 2 (black discs, n = 64) of sidering eight shape characters, four pilosity characters and Camponotus lateralis considering eight shape characters, head size. three pilosity characters and head size.

Tab. 3: Comparison of five diagnostic shape variables and a seta character between phenotype 2 workers of Campo- notus lateralis and three paratype workers from the holo- type nest of C. honaziensis after removal of allometric variance.

C. lateralis , C. honaziensis , phenotype 2 workers from ho- (n = 64) lotype nest (n = 3)

SL / CS 1.25 0.874 ± 0.020 0.974 ± 0.004 [0.822, 0.920] [0.970, 0.978]

SCI 1.25 1.023 ± 0.037 1.375 ± 0.012 [0.955, 1.122] [1.361, 1.384]

MW / CS 1.25 0.709 ± 0.015 0.754 ± 0.008 [0.669, 0.745] [0.749, 0.763] Fig. 5: Petiole scale in frontal view of phenotype 1 (Com- pacta morph, right) and phenotype 2 (normal morph, left) PRW / CS 0.252 ± 0.017 0.313 ± 0.005 1.25 of Lasius umbratus . Note the larger width and lower [0.218, 0.291] [0.308, 0.317] height of petiole scale and longer setae in the Compacta PRL / CS 1.25 0.326 ± 0.018 0.386 ± 0.013 morph. [0.272, 0.371] [0.377, 0.400] ral interspecific separations between related species in the nSc 1.25 8.51 ± 2.06 [4.0, 1.10 ± 1.35 subgenus Chthonolasius where error rates of up to 10% 12.3] [0.0, 2.6] may occur on worker individual level (B. Seifert, unpubl.). A proportion of 18.3% mixed nests is a very high value I conclude that phenotype 1 and 2 are the expression of a for an average within-nest sample size of only 3.0. Intra- panmictic, conspecific gene pool. specific dimorphism is by far the most probable explana- tion in this case indicated by much lower X2 values com- Case 2: Intraspecific shape-setae-pubescence pared to the three scenarios for heterospecificity. Pheno- dimorphism in Lasius umbratus type 1 differs from phenotype 2 in particular by a shorter The LDA confirmed the PCA clustering (Fig. 4) in 346 scape, reduced ratios of the maximum vs. minimum dia- individuals by 100%. The recognition rate in the re-clas- meters of scape and hind tibia, a smaller height of petiole sified sample was 97.4% for posterior probabilities of p scale, more dense pubescence on frontal head and gaster > 0.95. The discriminant values were separated by a gap tergites, a larger width of base and crest of petiole scale and were -2.314 ± 1.164 [-5.45, -0.37] in 57 workers of and longer setae on dorsal face of first gaster tergite. Fig- phenotype 1 and 2.865 ± 0.966 [0.37, 5.32] in 289 work- ure 5 shows typical petiole scales of both morphs. I name ers of phenotype 2. Most remarkably, the separation of this syndrome herewith "Compacta morph" due to the re- these intraspecific morphs was much stronger than seve- duced slenderness of several body parts, the more cylin-

52

Fig. 6 - 7: Head of Formica lugubris , (6) the Hippie morph and (7) the normal morph. drical appendage diameters and the denser pubescence. It should be mentioned in this context that I intended to de- scribe the Compacta morph as species in about 2006 hav- ing at hand in that time two pure nest samples of this well- different ant. Hastily translating this strong impression into a taxonomic act would have meant the production of a synonym. There is no clear argumentation to exclude environ- mental modification to have caused this dimorphism and we have to leave this question open. The Compacta syn- drome probably occurs also in the related Lasius distin- guendus (EMERY , 1916), but the number of reliably deter- mined samples available for an analysis was too low due to unsolved discrimination of workers from Lasius balca- nicus SEIFERT , 1982. A variant of the Compacta syndrome seems to represent the morph B of Cardiocondyla mauri- tanica FOREL , 1890 which is distinguished from the nor- mal morph by significantly reduced head length and shorter postocular head, shorter spines, more thickset petiole and postpetiole and longer gastral pubescence (SEIFERT 2003b). Case 3: intraspecific shape-setae dimorphism in Fig. 8: Principal component analysis of phenotype 1 (Hip- Formica lugubris pie morph, black discs, n = 108) and phenotype 2 (normal morph, grey squares, n = 369) of Fennoscandian Formica A PCA considering head size and the two most diagno- lugubris considering two pilosity characters and head size. stic characters – number of setae on dorsal plane of scape and on rear margin of vertex – separated the phenotypes a very strong pilosity. It differs from the normal morph by less clearly than in the previous cases. A LDA consid- longer and more numerous setae on dorsum of scape, pos- ering 11 characters confirmed the PCA clustering (Fig. 8) terior head margin and metapleuron. These characters are in 477 individuals by 99.6%. The recognition rate in the correlated with a more elongated head as well as a longer re-classified sample was 91.4% for posterior probabilities and narrower scape (Figs. 6, 7). The Hippie morph was of p > 0.95. A ratio of 14.9 % mixed nests is a significant supposed in the 1980s to represent a new Fennoscandian number for a mean within-nest sample size of 6.2 (Tab. 2). wood ant species (C.A. Collingwood, pers. comm.). The observed nest-type frequencies for the sympatric area in Sweden and Finland are most similar to the prediction Case 4: intraspecific shape-pubescence dimorphism in Cardiocondyla elegans for monogenic dimorphism. The next similar scenario, though having a clearly larger X2 value, is parabiosis. An NC-Ward clustering, which operates on the nest sample lev- observation sample of a real dimorphism case would show el, showed two clear clusters which provided the hypotheses some trend to a nest-type distribution of the parabiosis sce- for running an LDA on worker individual basis. The clas- nario if the panmixis condition is incompletely fulfilled. sification of this initial LDA served as hypothesis for the Maybe this is the case here. final LDA which indicated five mixed nests on the p > Phenotype 1 has been described by SEIFERT (2003a) as 0.95 level (Fig. 9, Tab. 2). These are 10.4% mixed nests the "Hippie" morph of the Fennoscandian population of within a sample of 48 nests. This figure is very high Formica lugubris. The Hippie morph is characterized by considering that mean within-nest sample size was only

53

Fig. 9: NC-Ward clustering of intraspe- cific dimorphism in Cardiocondyla ele- gans . Nests containing both phenotypes are pronounced by red label texts.

2.44 – a reduced chance to detect mixed nests. Among the ple level must not necessarily indicate two species, that four scenarios there is the highest similarity of the ob- mixed nests may be hidden within the clusters and that a served nest-type frequencies with the prediction for di- strong violation of the panmixis condition increases the morphism ( X2 3.43) but very close to this is the prediction probability of confusing parabiosis and dimorphism. for parabiosis ( X2 4.13) whereas both social parasitism sce- Case 5: the cryptic species Formica pressilabris and narios are clearly rejected. The biology of Cardiocondyla elegans is well known and just those populations being F. foreli the basis of this study have been extensively examined (LA- The cryptic species Formica pressilabris and F. foreli have FRÉCHOUX & al. 1999, 2000; LENOIR 2006, LENOIR & al. been separated by phenotype throughout their large Palae- 2007, MERCIER & al. 2007). Cardiocondyla elegans is a arctic ranges on the basis of nest sample means which is strongly thermophilous ant, typically occurring and being supported by data on ecology and altitudinal distribution most abundant on open riverine sand-gravel banks with in Central Europe (SEIFERT 2000). very sparse herb layer. At French rivers it is a pioneer Heterospecificity has been recently confirmed by NC species and indicative for the first stage of stabilization of clustering for almost the same data set used here: the error sand banks. It is in densely populated patches best con- relative to the controlling LDA was 1.7% in NC-Ward and sidered as polydomous-oligogynous on the colony level 0.0% NC-k-means clustering (SEIFERT & al. 2014b). In the with a high ratio of inbreeding: 70.4% of the copulations material investigated here, NC-k-Means clustering, which of winged gynes are with a brother (LENOIR & al. 2007). classifies on the nest sample level, provided the hypothesis It is clear from these studies that the phenotypes distin- for the initial LDA that classified (phenotyped) individu- guished here do not represent different species. Further- al workers. The phenotyping of this initial LDA served as more, it is most probable that the insular distribution and hypothesis for the final LDA which indicated 7 mixed nests rarity of habitats in connection with strong inbreeding on the p > 0.95 level (Tab. 2). These 3.4% mixed nests should result in considerable deviations from the panmixis within a sample of 206 nests from the sympatric zone are condition required for running the dimorphism model. The not a high value but may be understood as a warning sig- two morphs can be termed as slender and thick morphs. nal possibly indicating intraspecific dimorphism. Among Compared to the slender morph, the thick morph is a syn- the four scenarios considered here, parabiosis is clearly the drome of larger absolute size, relatively higher and wider most probable one ( X2 3.14) whereas all other scenarios petiole and a more dense pubescence of first gaster ter- including dimorphism ( X2 41.74) are extremely unlikely. gite. The example is instructive. It shows that a very clear These data show that the phenotypes of Formica pressi- clustering by an exploratory data analysis on the nest sam- labris and F. foreli represent clearly separate species. The

54 supposed intranidal phenotype mixtures might either in- characters. I used the nest-sample level classification of dicate occasional local hybridization, temporary parabiosis NC-k-means as hypothesis for the initial LDA to pheno- and / or a weakness of the parsimonious 6-character mor- type the individual workers but considered all 17 charac- phological identification system. ters. The phenotyping of this initial LDA served as hypo- thesis for the final LDA which indicated 1 mixed nest on Case 6: the cryptic species Temnothorax crassispinus the p > 0.95 level (Tab. 2). This means 2.3% of mixed and T. crasecundus nests within a sample of 44 nests from the sympatric zone. A separate species identity of the cryptic species Temno- Among the four scenarios considered here, parabiosis is thorax crassispinus and T. crasecundus has been shown clearly the most probable one ( X2 0.306) whereas all other 2 by SEIFERT & CSŐSZ (2015) who also rejected intraspecific scenarios including dimorphism ( X 8.52) are very unlike- dimorphism without providing an elaborated test system. ly. It was not confirmed by the collectors if the single The error of exploratory data analyses relative to the con- mixed sample was really collected from the same nest. If trolling LDA classification was 1.5% in NC-Ward and 2.0% so, the only explanations are a temporal parabiosis or a in NC-k-means clustering. I investigated here the same phenotyping error. data set as SEIFERT & CSŐSZ (2015) and used the nest- Case 9: the related species Myrmica scabrinodis and sample level classification of NC-Ward clustering as hypo- thesis for the initial LDA that classified (phenotyped) indi- M. vandeli vidual workers. The phenotyping of this initial LDA served There are no NUMOBAT data available for the 3763 nest as hypothesis for the final LDA which indicated two mixed samples investigated but only subjective determination nests on the p > 0.95 level (Tab. 2). These 2.3% mixed of about ten workers per nest by simple eye inspection. nests within a sample of 84 nests from the sympatric zone Apart from difficulties with the extremely rare interspe- are below the critical threshold. Among the four scenari- cific hybrids (BAGHERIAN YAZDI & al. 2012), Myrmica os considered here parabiosis is clearly the most probable scabrinodis and M. vandeli are well-separable by experi- (X2 0.631) whereas all other scenarios including dimorph- enced researchers on a subjective basis and the recogni- ism ( X2 16.28) are very unlikely. The supposed intranidal tion rate should have been about 90% during processing phenotype mixtures are either short-term temporary para- of such masses of specimens in this case. W. Münch bioses or classification errors. found in Baden-Württemberg 225 pure nest samples of M. vandeli , 38 mixed nests and 3500 pure nests of M. scabri- Case 7: the cryptic species Temnothorax luteus and nodis . This observation is inseparable from the prediction T. racovitzai 2 for parabiosis ( X 0.01) whereas all other scenarios have A separate species identity of the cryptic species Temno- a much higher test statistics (Tab. 2). Important from the thorax luteus and T. racovitzai has been shown by SEI - taxonomic point of view is the clear rejection of intra- 2 FERT & al. (2014a). They got an error of exploratory data specific dimorphism ( X 252.69) and the confirmation of analyses relative to the controlling LDA classification of heterospecificity. The prediction for temporary social para- both 0% in NC-Ward and NC-k-means clustering when sitism ranks next ( X2 48.88) and seems a possible option. the character set was reduced to the seven most diagnostic Myrmica vandeli has been supposed to be a temporary so- characters. SEIFERT & al. (2014a) found no mixed nests un- cial parasite of M. scabrinodis (SEIFERT 2007, RADCHENKO der this condition. I reinvestigated here exactly the same & ELMES 2010). The observations from Baden-Württem- material but considered the full set of 18 characters. The berg show a ratio of pure M. vandeli nests against mixed nest-sample level classification of SEIFERT & al. (2014a) vandeli -scabrinodis nests of about 6 : 1. The higher fre- was used as hypothesis for the initial LDA that pheno- quency of mixed nests compared to the figure of Lasius typed individual workers. The phenotyping of this initial (24 : 1) was explained by a hypothesized weaker tendency LDA served as hypothesis for the final LDA which indi- of the M. vandeli queen for early disabling or killing the cated one mixed nest on the p > 0.95 level (Tab. 2). These host queens (BAGHERIAN YAZDI & al. 2012). A clear bio- 1.9% mixed nests within a sample of 53 nests from the logical interpretation of the mixed nests can only be given sympatric zone are below the critical threshold. Among the by long-term observation of nest populations in laboratory. four scenarios considered here, parabiosis is clearly the 2 Error sources of the DIMORPH test and recommenda- most probable ( X 0.154) whereas all other scenarios in- 2 tions to avoid these cluding dimorphism ( X 14.21) are very unlikely. The sup- posed case of an intranidal phenotype mixture is probably Having a comparative look on the nine cases presented in a due to a phenotyping error. Table 2, the fixation of the relative nest-type frequencies in the three heterospecificity scenarios based on empirical Case 8: two cryptic species of the Pheidole pallidula data does not seem to be a substantial problem. These sce- complex narios have such different nest-type frequency distribu- The Westpalaearctic Pheidole pallidula complex contains tions that imprecise estimation of a particular nest-type four cryptic species which are distributed around the Me- frequency should not matter too much. Furthermore, a con- diterranean Basin (B. Seifert, unpubl.). Two of these, one fusion of these scenarios does not matter from a taxono- described and one still undescribed species, are most simi- mic point of view. lar and show a broad geographic range overlap in the Bal- More dangerous is obviously a violation of the pan- kans and Asia Minor. The error of exploratory data ana- mixis condition leading to a reduction of the mixed nest lyses relative to the controlling LDA classification was frequency in the observation sample as seen most clearly 2.8% in NC-Ward and 0% in NC-k-means clustering when in the Cardiocondyla elegans case. The nest-type frequency the character set was reduced to the seven most diagnostic distributions may then approach to the parabiosis condi-

55 tion. The same effect, namely a reduction of the proportion in just this rare phenomenon disproportionately. An inves- of mixed nests, would be caused by a geographic differ- tigator running the DIMORPH test should assess the re- entiation in phenotype frequencies. In all four presented levance of this error source. In the cases presented here, a cases of intraspecific dimorphism, parabiosis ranks on the sampling bias was excluded because it was impossible to second place in the range of scenarios. This suggests that determine the phenotypes in the field (cases 2 - 8). In the the ideal panmixis condition and / or homogenous geo- more easier separable phenotypes of case 1, each available graphic distribution of phenotypes is always violated to a sample was used without any bias because the material of certain degree (as one may expect). As a consequence, when both phenotypes was generally rare. In case 9 a sampling running the DIMORPH test, it is necessary for a researcher bias was also not given because the collector generally took to consider the mating scenarios in a species group (if each sample he found independent from his prejudice in known), the distribution type of a species caused by the the field. availability of adequate habitats and the geographic dis- A rare source of error occurs when the frequency of tribution of phenotypes as it appears from the data. The the recessive allele is very low. Assume an observation fact that dimorphism ranked first even in the Cardiocon- sample of 100 nests with zero pure nests of phenotype 1, dyla case indicates a certain robustness of the system pro- four nests with both phenotype 1 and 2, and 96 pure nests posed here. of phenotype 2, a recognition rate of 1.0 and a mean The presented genetic dimorphism model assumes the within-nest sample size of 10. Assuming the rarer pheno- presence of only a single and monoandrous queen. This type being homozygous recessive, the frequency of the re- should apply to the majority of ant species worldwide. cessive allele is then 0.10. The simulation of genetically Yet, polygyny is quite frequent in ants of the temperate mediated dimorphism will then predict the following fre- zone – as much as 28% of Central European species may quencies: 0.06% pure nests of the rarer phenotype 1, 1.92% occasionally or frequently form polygynous societies (SEI - nests with both phenotype 1 and 2 and 98.03% pure nests FERT 2007). Polyandry is much less abundant: mating num- of phenotype 2. This frequency distribution of nest types bers below 1.4 clearly predominate in ants on a world- of dimorphism is inseparable from the prediction for per- wide scale whereas mating numbers of up to 12 are only manent social parasitism that is 0 : 2 : 98. Assuming for known in very evolved societies of leaf-cutter, driver and the same scenario a recognition rate of 0.9 and a mean harvester ants (STRASSMAN 2001). A violation of the mono- within-nest sample size of 2.5, the prediction for dimorph- gyny-monoandry condition will act in just the opposite di- ism does not change strongly: 0.34% pure nests of the rarer rection compared to violating the panmixis and homogen- phenotype, 1.60% nests with both phenotype 1 and 2 and ous geographic distribution condition: An increased fre- 98.07% pure nests of phenotype 2. This frequency distri- quency of mixed nests and a reduced proportion of nests bution is also inseparable from the prediction for perma- with only recessive phenotypes. In other words, polygyny nent social parasitism. However, such cases should occur and polyandry are suspected to cause an erroneous rejec- rarely and there is only one procedure to minimize this tion of heterospecificity by the DIMORPH test. However, error: The taxonomist has to look if some characters of the as already explained above, polygyny is connected with supposed social parasite are in agreement with those norm- two opposing trends being likely to cause some kind of ally observed in that life form type. compensation on the metapopulation level. Polygyny will increase the chance that two phenotypes may occur within Conclusion the same nest but reduced exchange between viscose pop- The data confirm that the proposed DIMORPH test is a ulations of highly polygynous ants will simultaneously valuable taxonomic tool for eusocial insect taxonomists to violate the panmixis condition and reduce the number of assess the critical question if a phenotype constitutes a spe- mixed nests. Within the cases analysed here strong poly- cies or an intraspecific morph. Violation of the panmixis, gyny is given only in the case of Formica foreli vs. F. homogenous geographic distribution and monogyny / mono- pressilabris (SEIFERT 2007) where heterospecificity was andry conditions required for the test system may produce clearly confirmed. Fennoscandian Formica lugubris are errors and the investigator should consider this with care. monodomous and monogynous to weakly polygynous (PA- At least in the nine cases presented here, the DIMORPH MILO & al. 1994). Accordingly, the rejection of hetero- tests lead to reasonable conclusions. The value of the test specificity and acceptance of intraspecific dimorphism is becomes apparent when considering which types of error not likely of being caused by a violation of conditions re- matter within a taxonomic context. It seems also possible quired in the DIMORPH test. No disturbance of the hetero- that the apparently good performance, or robustness, is in specificity indication was also visible in Myrmica scabrino- some cases supported by compensating effects – e.g., a dis and M. vandeli which are weakly polygynous (SEIFERT violation of the panmixis condition reduces the number 2007). The remaining six cases refer to monogynous / of mixed nests whereas a simultaneous violation of the monoandrous to weakly polyandrous species and do not monogyny / monoandry condition increases the number of present a problem in this respect. mixed nests. The DIMORPH test has to be run in further A collecting bias by the taxonomist may also repre- cases before a reliable assessment of its performance can sent an error source. Due to limitations in working capaci- be done. ty and storage space, a taxonomist cannot afford collecting There is no doubt that only those rather few researchers and maintaining thousands of samples of an abundant spe- will use the DIMORPH test who "automatically" produce cies – maybe he stops after ten years when having gath- the data needed for the test because they are engaged in ered some 100 samples. If he suddenly recognizes a cer- Numeric Morphology-Based Alpha-Taxonomy. In other tain problem, such as a rare intraspecific dimorphism or a words, the test is a purposeful analysis of a byproduct of rare species, he may be biased to increase the sample size NUMOBAT research and researchers should seize this op-

56 portunity. Terminating a NUMOBAT study by a DIMORPH to zoological nomenclature (SCHLICK -STEINER & al. 2007, test constitutes a new form of multi-source taxonomy. An- STEINER & al. 2009). The frequent failure of genomics, other dimension is added to our system of vision: morphol- transcriptomics and proteomics in evaluation of type ma- ogy is projected on a series of templates indicating certain terial due to molecular degradation and / or curatorial pro- phenomena. It should become a routine process in ant tax- hibition of damage is reflected in a recent position paper onomy to assess if two separate clusters are heterospecific of about thirty German molecular taxonomists published or intraspecific. by the German National Academy of Sciences Leopoldina. There is also no doubt that ethological or genetic in- They proposed to degrade the taxonomic supremacy of vestigation may lead to much deeper insights in the nature primary type specimens (NATIONALE AKADEMIE DER WIS - of a phenomenon. Yet, those who emphasize the leading SENSCHAFTEN LEOPOLDINA 2014). position of genetics should also recognize the advantages Acknowledgments of non-destructive morphological phenotyping and its per- formance in delimiting cryptic species. NUMOBAT can I wish to thank Roland Schultz / Senckenberg Museum make use of the large worldwide collections of mounted of Natural History Görlitz for producing z-Stack photos, museum material or private collections of ants indepen- Wolfgang Münch / Tübingen for providing collection data dent from molecular degradation or curatorial ban of dam- on Myrmica, Celal Karaman / University of Edirne for do- age and can thus operate in fields where genetic investiga- nating paratypes of Camponotus honaziensis , Michel Cha- tion faces practical and logistic problems (FRANZ 2005, puisat / University of Lausanne for comments on gene- BROWER 2006, SCHLICK -STEINER & al. 2007, STEINER & tics of an earlier version of this manuscript, John M.C. al. 2009). The following excursus will explain this in more Hutchinson / Senckenberg Museum of Natural History Gör- detail. It is an enormous advantage of morphology-based litz and Markus Ritz / Görlitz for advice in the X² statis- taxonomy to make use of any collection material existing tics problem, and Pekka Pamilo / University of Helsinki in the natural history collections of the world beginning for very constructive criticism. with the type specimens of Linnaeus from 1758. An esti- mate of the costs to re-collect this material today in order References to obtain fresh material for DNA analysis would end in AKAIKE , H. 1973: Information theory and an extension of the billions of dollar. The logistic problem of contemporary maximum likelihood principle. In: PETROV , B.N. & CSAKI , F. morphology-based taxonomists is mainly arranging loans (Eds.): Proceedings of the Second International Symposium of already existing collection material and to a lesser degree on Information Theory. – Akademiai Kiado, Budapest, pp. of supplementing this by recent sampling. They have a 267-281. lower pressure for spending time, money, and manpower ANDRÉS , J.A. & CORDERO , A. 1999: The inheritance of female in organizing and carrying out expeditions to remote re- color morphs in the damselfly Ceriagrion tenellum (Odonata, Coenagrionidae). – Heredity 82: 328-335. gions of the globe several of which are developing today into dangerous areas. For example, my own NUMOBAT BAGHERIAN YAZDI , A., MÜNCH , W. & SEIFERT , B. 2012: A first demonstration of interspecific hybridization in Myrmica ants investigations included material collected by the expedi- by geometric morphometrics (Hymenoptera: Formicidae). – tions of Przewalski and Kozlov in North Tibet in 1876 - Myrmecological News 17: 121-131. 1902, of Forel in Libya in 1889 / 1893, or of Klapperich BARRION , A.A. & SAXENA , R.C. 1987: Inheritance of body color in Afghanistan in 1952 - 1953. Few scientists should seri- in the brown planthopper, Nilaparvata lugens . – Entomologia ously intend at the moment to collect in these regions. Experimentalis et Applicata 43: 267-270. Even if a research project does not require expeditions to BERNARD , F. 1968: Les fourmis d'Europe occidentale et septen- remote and dangerous parts of the globe, collecting fresh trionale. – Faune de l'Europe et du Bassin Méditerranéen 3, material of species with hidden nest sites and low popu- Masson, Paris, 411 pp. lation density (such as Lasius umbratus ) or of species be- BOOMSMA , J.J. & VAN DER HAVE , T.M. 1998: Queen mating and ing on the verge of extinction (such as Formica foreli and paternity variation in the ant Lasius niger . – Molecular Ecol- pressilabris in Central Europe) remains time consuming, ogy 7: 1709-1718. costly and is sometimes even prohibited by law. Nobody BOROWIEC , L. & SALATA , S. 2014: Redescription of Campo- can expect that collectings done by naturalists over 150 notus nitidescens FOREL , 1889, new status and notes on ants years can be repeated within the time frame of a recent from Kefalonia, Greece (Hymenoptera: Formicidae). – Genus research project of gene-based taxonomy. The recently en- 25: 499-517. acted Nagoya Protocol on Access and Benefit-Sharing ad- BROWER , A.V.Z. 2006: Problems with DNA barcodes for spe- ded further difficulties for collecting of specimens for gen- cies delimitation: "ten species" of Astraptes fulgerator reas- etic investigations. sessed (Lepidoptera: Hesperiidae). – Systematics and Biodi- versity 4: 127-132. The non-destructive investigation of type material is perhaps the most deciding advantage of morphology-based BURNHAM , K.P. & ANDERSON , D.R. 2002: Model selection and multimodel inference: a practical information-theoretic ap- taxonomy. Taking a small tissue sample for DNA analysis proach. – Springer, New York, NY, 488 pp. from the ball of toes of a monkey-eating eagle or from the BUSCHINGER , A. 2005: Experimental evidence for genetically skin of a quagga does not significantly affect the value of mediated queen polymorphism in the ant species Myrmecina the particular type specimen. However, if one requires to graminicola (Hymenoptera: Formicidae). – Entomologica Ge- take a DNA sample from the primary type specimen of a neralis 27: 185-200. small insect, responsibly thinking museum curators should CHAPUISAT , M., GOUDET , J. & KELLER , L. 1997: Microsatellites prohibit this damage. These restrictions result in a help- reveal high population viscosity and limited dispersal in the lessness of gene-based taxonomy in linking gene clusters ant Formica paralugubris . – Evolution 51: 475-482.

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