This study is based on an examination of ~ 570 specimens of Anoplistes halodendri minutus and A. kozlovi sensu Danilevsky and Smetana (2010) from the territory of Mongolia (Figs 1, 2, 3) as well as additional ~ 110 specimens of A. jacobsoni from southern Kazakhstan and ~ 20 specimens of A. halodendri halodendri from eastern Kazakhstan. The specimens are housed in the following collections: CCH, the collection of Carolus Holzschuh, Villach, Austria; CLKR, the collection of Lech Kruszelnicki, Siemianowice Śląskie, Poland; CWTS, the collection of Wojciech Szczepański, Siemianowice Śląskie, Poland; HNHM, the Hungarian Natural History Museum, Budapest, Hungary; MAS, the Institute of Biology, Mongolian Academy of Sciences, Ulan Bator, Mongolia; MIZ, Museum and Institute of Zoology, Polish Academy of Sciences, Łomna, Poland; NMP, the National Museum Prague, Prague, Czech Republic; USMB, the Upper Silesian Museum, Bytom, Poland; ZIN, the Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia.

The individuals that were used for the detailed morphological and molecular analyses were collected by the authors during entomological expeditions to Kazakhstan (2017) and Mongolia (2019). Moreover, additional fresh material for the analyses was collected in SE Mongolia by Badamnyambuu Iderzorig (National University of Mongolia, Ulan Bator, Mongolia) earlier in 2019.

Figure 1. 

Distribution of the two ecotypes of Anoplistes halodendri minutus in Mongolia and the adjacent territory (OpenStreetMap contributors). Regions “A” and “B” are presented in detail in the following figures.

Figure 2. 

Google’s close-up satellite image in a desert area in southern Mongolia (region “A” marked in Fig. 1) with the localities of the rock ecotype of Anoplistes halodendri minutus (Google Earth).

Figure 3. 

Google’s close-up satellite image in the northern steppe area in central Mongolia (region “B” marked in Fig. 1) with the localities of the sand (yellow spot) and rock (red spot) ecotypes of Anoplistes halodendri minutus (Google Earth).

Two nearly sympatric Anoplistes populations were found in one extensive site (LK1) in the environs of the Choiriin Bogd Mountain [Чойрын Богд Уул] (46.24, 108.77), approx. 30 km SEE of Choir [Чойр] (“purple star” in Fig. 1; Fig. 4) on 17 and 19 July and 1 August 2019. The first population, which was found in a small canyon and on rocky slopes of the surrounding mountains (plot 128 in Fig. 4; Fig. 5A, B), consisted nearly exclusively of individuals with an almost completely blackened elytra with two reddish spots in the basal part and a single stripe along each epipleuron (Fig. 5C, D), which corresponded to the habitus of A. halodendri minutus—a widely distributed subspecies in Mongolia. However, after leaving the rocky area and continuing our sampling towards the flat terrain with a gravelly-sand surface with no rocks (plot 129 in Fig. 4; Fig. 5E), we began to find almost exclusively individuals with a reduced main black spot and predominance of pale orange on the elytra (Fig. 5F, G), which seemed to correspond to A. kozlovi—another taxon of the genus that is widespread in southern Mongolia and northern China (Inner Mongolia). It is worth noting that while only a few (< 5%) individuals with a reduced black spot were found in the rocky part (plot 128) of the site (and only at lower elevations), in the second part of the site (plot 129), the other forms were slightly more numerous (~ 10%). An incidence of copulation between two different forms was observed in several pairs, mainly in the transitional zone (Fig. 5H). The detailed structure of the site with the approximate proportions of the individuals of both forms is presented in Fig. 4.

Figure 4. 

Detailed view of the main study site in the Choiriin Bogd Mountain (plots no. 128 and 129) in eastern Mongolia, where both ecotypes of Anoplistes halodendri minutus occur sympatrically (OpenStreetMap contributors).

Figure 5. 

Field photographs of imagines in nature and the habitats of Anoplistes halodendri minutus. A, B: view on rocky habitat. C, D: the rock ecotype of A. h. minutus. E: view on sandy/gravelly habitat. F, G: the sand ecotype of A. h. minutus from the Choiriin Bogd Mountain. H: view on transition zone from sandy to rock habitat.

All of the individuals were found sitting on Caragana Fabr. (Fabaceae) shrubs. Although the pea-shrubs that grow in both plots of the above-described site appeared somewhat different in their structure, it was determined by local botanists that the pea-shrub individuals within the entire locality belong to the same species—Caragana leucophloea Pojark (Fig. 6A). Additionally, a second species—Caragana bungei Ledeb. (Fig. 6B)—occurs in the rocky part of the site. However, the adults of Anoplistes in the stony habitat were commonly sitting on both Caragana species. The development of the larvae of A. halodendri minutus in the discussed plant species was confirmed by the literature data (Karpiński, Szczepański, Boldgiv et al. 2018). Therefore, the two Anoplistes forms that were observed seem to be ecologically associated with the same hosts and some of the differences in the C. leucophloea structure most likely result from growing on a different type of subsoil—rocky (Fig. 6C) and sandy/gravelly (Fig. 6D). Anoplistes jacobsoni, on the other hand, is most likely monophagous on Halimodendron halodendron (Pall.) Voss. (Fabaceae) (Karpiński, Szczepański, Plewa et al. 2018).

Figure 6. 

Field photographs of imagines in nature, host plants and habitats of Anoplistes halodendri minutus. A: Caragana leucophloea. B: Caragana bungee. C: C. leucophloea on rocky ground. D: C. leucophloea on sandy/gravelly ground. E, F: the sand ecotype of A. h. minutus from southern Mongolia. G, H: the desert habitats in southern Mongolia; (photos E–H by Badamnyambuu Iderzorig).

In order to confirm the relationship between the specific form and the type of environment, the habitus and habitat of the specimens from other regions of Mongolia (Figs 1, 2, 3) were analysed. First, we examined specimens of two populations (BI1 and BI2; Fig. 6E, F) that were recently collected in desert habitats (Fig. 6G, H) in the southern part of the country (Fig. 1: “framed triangles”) whose DNA sequences were also used for a molecular analysis. All of the specimens (N = 20) were almost uniform in their elytral pattern and bright colouration and corresponded to the habitus of A. kozlovi sensu Danilevsky and Smetana (2010) from the previously discussed locality. Subsequently, we studied additional museum and private specimens of numerous other populations in order to determine, where possible, the exact habitat in which the beetles were collected—either using precise geographical coordinates (when provided) or the detailed descriptions of the sites provided by Zoltán Kaszab in his expedition reports (Kaszab 1964, 1965, 1966, 1967, 1968, 1969). In some cases, there was only one type of habitat in a region, thus the precise localisation of the plot was not necessary. The obtained data confirmed a connection between the given form and one of the two habitat types.

An interpretation of the ecological field data suggests the ecotypic, rather than a specific differentiation.

Over 500 specimens of Anoplistes halodendri minutus (Fig. 7A–F) and A. kozlovi sensu Danilevsky and Smetana (2010) (Fig. 7G–L) from the territory of Mongolia were studied including more than 200 that were collected in the main site (Choiriin Bogd Mountain).

Figure 7. 

Habitus of some taxa of the Anoplistes halodendri species-group. A–M: Anoplistes halodendri minutus. A–F: rock ecotype; G–L: sand ecotype—Anoplistes kozlovi sensu Danilevsky and Smetana (2010). M: intermediate form. N, O: Anoplistes halodendri halodendri. P, Q: Anoplistes jacobsoni. A, C, E, G, I, K, M, N, P: males. B, D, F, H, J, L, O, Q: females. Scale bar: 5 mm.

Results of the detailed morphometric measurements of 100 individuals of both forms revealed no statistically significant differences in the body length. Total body length in the males ranged from 7.6 to 11.4 mm (“minutus”, N = 30) vs 7.9 to 12.5 mm (“kozlovi”, N = 30); in the females, it ranged from 8.6 to 12.8 mm (“minutus”, N = 20) vs 8.0 to 12.0 mm (“kozlovi”, N = 20); 25–75% quartiles practically overlapped between the examined specimens of the two forms (Fig. 8A, B). It is worth noting that the individuals that represented the southern populations of A. kozlovi sensu Danilevsky and Smetana (2010) (BI1 and BI2; Fig. 1: “framed triangles”) were relatively bigger than those from the Choiriin Bogd Mountain population (LK1). In a sample of 13 specimens, most were larger than 10 mm (mean for males: 11.25 mm, N = 8; for females: 11.2 mm, N = 5). Regarding the maximum value of the ratio of pronotal width and length, it was significantly smaller in the “kozlovi” form for both the males and females (Fig. 8C, D). However, this trait cannot be diagnostic as the values for most of the individuals of both subpopulations were in the same ranges. In the case of the elytral shape, although there were some differences in the maximum values for the males, the values for the females had a nearly total overlap (Fig. 8E, F). Similarly, when the ratios of the individual antennomeres to antennomere 1 were considered, no statistically significant differences were found (Fig. S1).

Figure 8. 

Measurements of the body lengths and pronotum and elytra shapes (ratio of its total length to its maximum width) in the males and females of Anoplistes halodendri minutus and Anoplistes kozlovi sensu Danilevsky and Smetana (2010).

Comparative analysis of the pubescence density and length using SEM technology indicated that within both forms, densely pubescent and almost completely hairless individuals (considering the elytra and pronotum) occurred (Fig. 9). This applied to the newly hatched individuals, which were collected in the same place and at the same time and did not appear to be connected to “wiping off” of the beetles, but rather to a natural individual variability. A closer examination of the SEM images also enabled it to be confirmed with certainty that there were no differences in the sculpture and punctation of the pronotum and elytra.

Figure 9. 

SEM images of the pronotum and elytra of Anoplistes halodendri minutus presenting the differences in pubescence density. A–D: rock ecotype. E–H: sand ecotype.

Clearly, the best trait for distinguishing both ecotypes is the shape and the range of the elytral spot, and, to a lesser extent, the colouration of their lighter part. The full range of the variability of these traits for both forms is presented (Fig. 10). In both ecotypes, there were patterns with or without bright spots in the basal part of the elytra, however, generally, the main black elytral spot was clearly narrower and spread differently in the “kozlovi” compared to the “minutus” (Fig. 10A vs B). The colouration of the lighter part of the elytra was almost always pale orange in the “kozlovi” and usually reddish in the “minutus”.

Figure 10. 

Variability of the elytral pattern in the specimens of Anoplistes halodendri minutus. A: rock ecotype. B: sand ecotype.

An examination of the lateral lobes of the tegmen revealed no differences between the discussed ecotypes (Fig. 11). Generally, these structures had a relatively high variability and numerous specimens within both environmental forms had an almost identical structure (e.g. Fig. 11A vs G). Additionally, the parameters of the seemingly very close species of this group—A. jacobsoni (Fig. 11K–O) were also analysed. Although most of the individuals appeared to have distinctly different lateral lobes, after studying a sufficiently large series, it was found that some of them had structures that were very close to those of A. halodendri (Fig. 11O vs C, respectively). This highlights the need for future studies to examine the genitalia of many specimens before drawing conclusions about the separateness of two entities. Notwithstanding, there was a clear stronger dissimilarity of A. jacobsoni.

Figure 11. 

Variability of the lateral lobes of the tegmen in the Anoplistes halodendri species-group. A–J: Anoplistes halodendri minutus. A–E: rock ecotype. F–J: sand ecotype—Anoplistes kozlovi sensu Danilevsky and Smetana (2010). K–O: Anoplistes jacobsoni.

The shape of the median lobe (Fig. S2) appears to be a taxonomically uninformative character owing to its variability between taxa, being similar to the genus Ropalopus Mulsant, 1839 (Callidiini) of the same subfamily (Karpiński et al. 2020). More importantly, an examination of the endophallic structures did not reveal any stable differences between A. halodendri minutus and A. kozlovi sensu Danilevsky and Smetana (2010). Some differences between these two forms (e.g. in the length of the baso-ventral tubercles, in the shape of the apical tubercles and the chambers) were weak and not stable and they result from individual variability (Fig. 12). Regarding A. jacobsoni, the most distinct difference was the shape of the BLV-sclerites. While in A. halodendri the lateral arc of the BLV-sclerites was crescent- (or C-) shaped and moderately short, in A. jacobsoni this structure was longer and comma-shaped.

A detailed examination of the morphology of the specimens supported the ecological data in concluding that the two discussed entities seem to represent only ecologically adapted forms and therefore should not be treated as separate species.

Figure 12. 

Variability of the endophallic structures in the Anoplistes halodendri species-group. A–C: Anoplistes jacobsoni, with the terminology used in this paper. D–K: Anoplistes halodendri minutus. D–G: rock ecotype, two different specimens. H–K: sand ecotype—Anoplistes kozlovi sensu Danilevsky and Smetana (2010), two different specimens. A, D, E, H, I: lateral view. B, F, J: ventral view. C, G, K: dorsal view.

It is common among the sedentary species that occur in isolated populations to exhibit a strong intraspecific genetic differentiation, which is driven either by geographical isolation or by a local ecological adaptation to specific environmental conditions. While the first factor has frequently been studied, the genetic effects from a local ecological adaptation are much less recognised (Schluter 2009; Lechner et al. 2015). Ecotypes are intermediate stages in ecological speciation. They are groups of populations within one species that are distinguishable due to their adaptation to local environmental conditions (Lowry 2012). They increase the distribution area of a species by adjusting to diverse habitats. Such an adaptation, which depends on various variables (e.g. the type of response, trait, population, environmental conditions), can take many shapes and can occur at different speeds (Kristensen et al. 2018). Adjusting to different conditions via divergent natural selection can generate phenotypic and genetic differences in the ecologically important characters between local populations of the same species. If the gene flow is sufficiently reduced, these eventually may lead to the formation of new species (Schluter 2000; Nosil 2012). In that case, however, before the entire process is completed, some characteristic stages occur that can be identified.

Kruckeberg (1986) proposed a model for the evolution of a plant endemic in which he identified several such steps. It seems that a slightly modified model could also be used for animal ecotypes:

0 Some form of preadaptation in order to develop a tolerance to a specific environmental variable exists in the normal population of a species. This provides the conditions that permit the evolution of an ecotype that is better adapted to the new conditions;

1. Disruptive selection causes the separation of the species into different gene pools. This is the formation of an ecotype;
2. Further genetic divergence in the phenotypic and functional traits occurs within the ecotype;
3. Isolation between the two ecological forms becomes genetically fixed, such that the two gene pools are unable to exchange genes;
4. Further divergences of the incipient species occur, which is “put in motion by the initial genetic discontinuity”.

However, according to Pandeya and Lieth (1993), the origin of ecotypes remains a difficult issue in population ecology as genetic variability, which is the basis for the formation of an ecotype, will constantly undergo natural selection before it is established. A mutation and recombination of genes must be the residual effect of the action, interaction and reaction between the environment and an organism and cannot be spontaneous. In other words, it is the environment that is a dominating factor in determining population differences.

It can be problematic to recognise and classify intraspecific forms and even more so to further distinguish possible ecotypes and subspecies, especially since both categories can occur within one species, as, for instance, is the case with the reindeer Rangifer tarandus (Linnaeus, 1758) (Gravlund et al. 1998).

A subspecies rank is commonly recommended to be used to recognise geographic distinctiveness. This term usually refers to one of two or more populations of a species that live in different regions of the species’ range and that differ from one another by a relatively weak morphological differentiation. The main criterion for recognising two distinct populations as subspecies, rather than as separate species, is their ability to interbreed without a fitness penalty. In natural conditions, subspecies do not interbreed due to their geographic isolation or sexual selection. A subspecific status should be considered when geographically separate populations of a species exhibit recognisable and predominantly stable phenotypic trait values. In other words, a subspecies is a recognised local variant of a species (Russell et al. 2011).

Conversely, an ecotype status is frequently assigned if a given form occurs throughout the geographic range of a species. Additionally, an ecotype is a variant in which the phenotypic differences are too few, too subtle or too unstable to warrant being classified as a subspecies. No taxonomic rank can be determined due to a lack of sterility barriers at this stage. Ecotypic variations can occur in the same geographic region where distinct types of habitats such as sand dunes and rocky slopes provide ecological niches. In the event that similar niches occur in widely separated places, similar ecotypic forms that are adapted to the effects of a very specific local environment can occur independently (Langerhans et al. 2007; Riesch et al. 2013).

Thus, the main difference between these two terms is that a subspecies can exist across a number of different habitats and would usually be limited to a restricted part of a species’ range while ecotypes occur throughout the geographic range of a species in similar ecological niches.

As has been justified above, Anoplistes halodendri in Mongolia, northern China (Inner Mongolia) and southern Siberia has developed into two distinct ecotypes. Populations of sand and rock ecotypes of A. halodendri minutus are found throughout the geographic range of this taxon. The same pattern was documented in the case of lizards of the Eremias multiocellata—E. przewalskii species complex, which are also found in the region (Orlova et al. 2017). Therefore, the synonymisation that was proposed by Namhaidorzh (1972): Cerambyx halodendri Pallas, 1773 = Cerambyx ephippium Steven and Dalman, 1817 = Anoplistes minutus Hammarström, 1892 = Asias kozlovi Semenov and Znoiko, 1934, is partially correct. Despite the results of the COI analysis, it is too early for us to comment on the synonymisation with Anoplistes halodendri halodendri (Pallas, 1773) and, in particular, we do not agree with the synonymy of Anoplistes halodendri ephippium (Steven and Dalman, 1817), which is mainly distributed in the southern part of European Russia. While the taxonomic position of the latter requires further research (in prep.), the preliminary results based on mitochondrial DNA support its dissimilarity. Although Danilevsky (2019) also recognised this extensive synonymisation as incorrect, he rejected the synonymy of A. kozlovi as well, claiming that it is definitely a separate species. The differences between these two taxa that he identified were disproportions in the elytral and pronotal pubescence, pronotal punctation and body size. In the presented study, we clarified that any differences in these traits result from individual variability and cannot serve as stable diagnostic characters. Furthermore, he based his conclusions only on the study of the series of A. kozlovi sensu Danilevsky and Smetana (2010) from Arkhangai Province (47.33, 103.68). Representatives of the same population were also examined by us and although they indeed stand out with considerable body size, numerous individuals of this sand ecotype are still smaller than the regular individuals of the rock form.

To conclude, the detailed morphological analysis, in particular regarding the endophallic structures, as well as the lack of clear patterns in the nuclear trees and relatively very low COI genetic distances (especially considering much higher genetic distances for morphologically and ecologically close A. jacobsoni) strongly indicate the cohesiveness of A. h. minutus including A. kozlovi and the need for synonymization.

It is worth mentioning that only one case of ecotypic variation has been documented in the family Cerambycidae to date. The yellow spotted longicorn beetle Psacothea hilaris (Pascoe, 1857) (Lamiinae: Lamiini) was divided into several subspecies, which are distributed mainly in Japan (Danilevsky and Smetana 2010). Psacothea hilaris hilaris has developed two ecotypes in Honshu, which, besides their elytral pattern, primarily differ in their larval biology, which do or do not diapause (Sakakibara and Kawakami 1992; Sakakibara 1995a,b). Thus, the reported environmental adaptation of Anoplistes halodendri is the second documented ecotypic speciation in this beetle family. Considering the entire superfamily Chrysomeloidea, several examples of ecotypes were also documented; mostly, however, in the family Chrysomelidae and for the species of high economic importance: Leptinotarsa decemlineata (Say, 1824) (Hsiao 1978), Galerucella aquatica (Geoffroy, 1785) (Lohse 1989), Galerucella lineola (Fabricius, 1781) (Ikonen et al., 2003), and Oreina speciosissima (Scopoli, 1763) (Borer et al. 2011).

The beginnings of the differentiation of A. halodendri in the territory of today’s Mongolia most likely can be traced back to the period of the intensification of aridification in the region and the formation of the Gobi Desert ~ 24 to 2.6 Ma (Lu et al. 2019). As there were probably no significant geographical barriers for flying insects in the region at that time and considering the almost sympatric distribution of the populations of both ecotypes in the Choiriin Bogd Mountain and some other known localities, it seems rational to assume that parapatric speciation is ongoing here. This model of speciation has already been documented in Cerambycidae for both the flying (Karpiński et al. 2020) and flightless taxa (Nakamine and Takeda 2008). Furthermore, evidence for parapatric speciation in the case of sympatrically distributed ecotypes was provided by Fisher-Reid et al. (2013) in a study on the salamanders of the genus Plethodon Tschudi, 1838. Their results support the idea that the spatial segregation of sympatric ecotypes may play an important part in this speciation model. It is worth noting, however, that in the presented case of ecotypic speciation in the genus Anoplistes, no switch to a new host plant was observed, unlike in several documented cases in some groups of insects (e.g. Sandoval and Nosil 2005; Nosil et al. 2006).

Given some of the rather stable differences in the phenotype of distant sand form populations, we consider it likely that similar ecotypes arose independently in different parts of the species’ range under parallel speciation. Individuals from the sand dunes in the northernmost part of the range differ from those from gravel desert in southern Mongolia and those from the Choiriin Bogd Mountain site in the steppe region (Fig. 7G, H vs I, J vs K, L, respectively)—although all of these populations are somewhat different, some specimens are sometimes indistinguishable from one other. Still, however, the rock ecotype is more homogeneous in phenotype throughout its range. This may lead to the conclusion that some part of the different populations of the rock ecotype had begun to transform in response to the increasing desertification. The first mentioned form remained on the stony hills (Fig. 2) within its entire range, while the latter began to adapt to new habitats simultaneously in many places. It has already been proven that common environmental factors repeatedly lead to the evolution of similar phenotypic forms in independent geographic localities under parallel speciation (Schluter and Nagel 1995; Johannesson 2001; Nosil et al. 2002; Schluter 2009). Multiple origins were supported in several studies, for instance, in the populations of threespine sticklebacks (Rundle et al. 2000, Taylor and McPhail 2000, McKinnon et al. 2004), Timema walking sticks (Nosil et al. 2008), Littorina marine snails (Butlin et al. 2008), and Gambusia mosquito fishes (Langerhans et al. 2007). Moreover, it was shown in these studies that individuals of the same ecotype are more likely to mate, regardless of relatedness as indicated by phylogenetic affinity.

The results of the COI analysis seem to confirm this scenario to a certain extent as the individuals of the rock ecotype from the Choiriin Bogd Mountain population turned out to be identical to the individuals of the very remote Kazakh population that share the same phenotype, while the individuals of the sand ecotype from two different regions in Mongolia, probably due to some assortative mating connecting populations in desertified habitats, were closer with each other (median, 0.18%) than with the neighboring population of the opposite ecotype (median, 0.71%). Still, however, when analysing the genetic distances more carefully, “minutus” revealed a closer relationship to the adjacent individuals of “kozlovi” (median, 0.71%) than to “kozlovi” from the southern populations (median, 0.89%).

Genetic data holistically supports the proposal that ecotypes can sympatrically form and develop a diagnosable morphology as a step towards reproductive isolation. The nuclear data show species level breaks in other taxa of Anoplistes yet there is no shared polymorphism in each of the putative ecotypes. The mitochondrial data, which evolve with a smaller effective population size than nuclear genes, indicate some isolation between ecotypes. A continuation of the observed population dynamics will be necessary for evolution beyond this incipient stage in the speciation process.

The differences in elytral colour pattern are probably correlated with the soil substrate. It seems opportunistic in more desertified habitats to develop lighter colored (pale orange) elytra with a narrow black stripe (most likely imitating the shadow of a peashrub stem). Colours in this pattern visibly better camouflage the beetle against the sand/gravel substrate and shrub structure, compared to almost entirely black elytra with two red spots, which in turn may work much better in spatially diversified rocky habitats with numerous objects that create varied shadows. Elytral and pronotal colouration resembling grass shadows on the ground is found in many species of the genus Eodorcadion (Cerambycidae: Lamiinae) that are widely distributed in this region. Most of the species have black elytra with a few wide stripes formed of white pubescence on each elytron. Such a contrasting colouration seems to be poor camouflage in desert and steppe habitats, however upon careful observation of these beetles, it becomes clear how effectively this pattern resembles shadows of Stipa grasses, especially in full sun.

It seems that the two discussed ecological forms of Anoplistes have not yet developed barriers to gene flow (reproductive isolation) as we observed a few mating couples in their contact zone. There are also no differences in endophallic structures, which makes us think that mating may be successful and not futile as is often the case between co-occurring anthophilous cerambycid species. Individuals from two adjacent habitats can meet and occasionally mate, however the total number of such mixed couples constitutes only a small percentage of the populations. Moreover, such a situation is only possible where these distinct habitats border each other, while the greater part of the species’ area is either flat sandy/gravelly plains or grassy steppes with rocky hills and the mountain regions. Furthermore, in addition to the above-mentioned fact that individuals of different ecotypes are less likely to mate, it seems reasonable that “alien” individuals die faster in the ecotone, and especially in the opposite habitat, as they are more easily spotted by predators, making these copulations even more sporadic and any offspring less fit. This is one of the basic mechanisms in parapatric speciation. In this mode, divergence may happen because of reduced gene flow within the population and varying selective pressures across the population’s range.

While Anoplistes was recovered as monophyletic in both the BI and ML analyses for all three genes combined with very strong or strong support (respectively), interestingly, Amarysius and Purpuricenus were rendered as paraphyletic. Although the representation of these genera (mainly for Purpuricenus as Amarysius includes only five species) might not be comprehensive enough to judge on divisions within this tribe, our results are consistent with those of the latest study in the subfamily Cerambycinae (Lee and Lee 2020). In our analyses, Amarysius altajensis was placed (albeit with weak or moderate support) in one clade with all of the Anoplistes taxa, which is in the position of a sister group to Amarysius sanguinipennis. Low interspecific differences in the endophallic structures in this group indicate that the clades of Anoplistes and Amarysius are evolutionarily relatively young.

Within Purpuricenus, European P. kaehleri was placed (with very strong support) into one clade with the Asian genera Anoplistes and Amarysius as a sister group to the clade of the Asian Purpuricenus (P. sideriger and P. temminckii), which in turn is in the position of a sister group to European P. desfontainii. In the aforementioned study, Purpuricenus lituratus Ganglbauer, 1886 (not tested by us) revealed the paraphyly of the genus (Lee and Lee 2020).

Surprisingly, A. jacobsoni revealed a strong divergence and long genetic separation from the morphologically very similar A. halodendri, though it was even considered to be a synonym of the latter by Kostin (1974). Therefore, its species status has been confirmed and the possibility of convergent evolution in morphological traits needs to be carefully examined, especially since the A. jacobsoni clade was the sister group to the clade of A. halodendri and A. galusoi. The latter species differs significantly in its morphology and bionomy and seems to belong to a different lineage with distinct ecological associations with another group of host plants—Ephedra L. (Ephedraceae). Further taxonomic work on the phylogeny of this genus is needed and requires the use of both morphological and molecular approaches.