New insights from biogeography, morphology and molecular biology: the species status of Acalles temperei Péricart, 1987 and Kyklioacalles navieresi (Boheman, 1837)
(Curculionidae: Cryptorhynchinae) 
by 
Peter E. Stüben, Mönchengladbach 
& 
Jonas J. Astrin, Bonn 
(14 photographies, 2 plates, 1 diagram and 8 distribution maps)

[Stüben] 

Abstract 

Biogeographical and morphological arguments are presented that suggest the following synonymization: Acalles parvulus Boheman, 1837 = Acalles temperei Péricart, 1987. Comparing the aedeagi along a transect from Mont Pilat (Dep. Loire) to Mont Saint-Martin North of Grenoble (Dep. Isère), a cline becomes apparent that raises doubts about the species status of Acalles temperei. These results, obtained by comparative phenotypic examination, are corroborated in a preliminary set of molecular genetic analyses. The latter either hint the existence of a hybrid zone or indicate that Acalles parvulus and Acalles temperei constitute a single, geographically structured species.

The molecular results for Kyklioacalles roboris (Curtis, 1834) and the recently resynonymized species Kyklioacalles navieresi (Boheman, 1837) are very different. DNA sequence analysis of the mitochondrial CO1 and 16S genes and of the nuclear 28S gene showed that Kyklioacalles navieresi and Kyklioacalles roboris likely represent two individual, but closely related species. 

Key words 

Coleoptera, Curculionidae, Cryptorhynchinae, Kyklioacalles, Acalles, integrative taxonomy, morphology, DNA taxonomy, barcoding, mitochondrial, nuclear, 16S rRNA, COI, CO1, 28S rRNA, ecology, distribution, biogeography, West-Palearctic, France. 

1. Introduction 

The present contribution on biogeographical, morphological and molecular analyses concerning the species status of Acalles temperei Péricart, 1987 and Kyklioacalles navieresi (Boheman, 1837) is a new outset in a threefold way: 

1. The CURCULIO-Institute (CURCI; Germany: Mönchengladbach) and the Zoological Research Museum Alexander Koenig (ZFMK; Germany: Bonn) join forces and practically implement interinstitutional research. In close cooperation, both sides provide each other with facilities and relevant infrastructure in order to shoulder a task hard to address alone.

2. A modern integrative, interdisciplinary taxonomy combines knowledge and expertise of morphologists and molecular systematists, linking phenotypical analysis with DNA sequence analysis and - in special cases - with crossbreeding experiments. 

3. In many well-investigated groups of western Palearctic Curculionidae we have long since reached the 'morphological limits' of our efforts in differential diagnosis. The exclusively phenotypic and morphological, extremely creative treatment of tiny character details in systematics often boils down to an artistic way of self-affirmation. 

Of course, species differentiation cannot do without morphological studies on complex and visually easily apprehensible phenotypic structures or without observations on ecology or reproductive biology. However in cryptic taxa (as the here discussed species pairs), morphology does not suffice to reconstruct the status and phylogeny of species. Also necessary is a specialist-developed but generally applicable, fast routine identification procedure, especially for larvae or females, which so far were often impossible to identify in Cryptorhynchinae. To this purpose, DNA sequence analysis is a highly convenient complementary method as it provides a nearly unlimited number of characters. Individually, these characters are less complex and hence of lower information content than morphological characters [Wägele 2001: 117], nevertheless they can always be easily quantified, reproduced, are discrete, and can be homologized over long phylogenetic distances [Astrin et al., in press]. Finally, DNA sequencing is increasingly getting cheaper and more and more steps are automatized. 

2. Acalles parvulus Boheman, 1837 and
Acalles temperei Péricart, 1987 

2.1 Taxonomy, morphology and biogeography 

In his first description of Acalles temperei, Péricart points out that the form of the aedeagus is the most significant character distinguishing this species from the sibling species Acalles parvulus Boheman, 1837. As a matter of fact both taxa are undistinguishable as far as ectoskeletal morphology is concerned: [Fig. W33.1][Fig. W33.2]. However in the ventral view, the median lobus of the aedeagus of Acalles parvulus is acutely rounded, whereas in Acalles temperei it forms an obtuse-angled tip. In the lateral view, this tip seems to be somewhat separated from the slightly broader median lobus: [Fig. W33.3]. This character seemed to be sufficient for Péricart to distinguish between both taxa definitely. [Péricart 1987] 

In fact, there exist specimens of Acalles temperei from the locus typicus (Pyrénées-Orientales), from south-eastern France (Rhône-Alpes), from western Switzerland (vicinity of Genève) and western Italy (Piemonte) featuring the typical aedeagus characters. In comparison to the wide area of distribution of Acalles parvulus from the Rhineland (Germany) to Sicily (Italy) and from eastern Austria (vicinity of Graz) to southern France (Pyrénées-Atlantiques), the distribution of Acalles temperei is 'local and disjunct'. [Fig. W33.4]. We do not know any other European (flightless) species of Acalles, Dichromacalles, Kyklioacalles, Onyxacalles or Echinodera showing such a disjunct distribution pattern. Between the extremely isolated populations in southern France (Pyrénées-Orientales) and the localities in the vicinity of Grenoble (Rhône-Alpes) there are numerous populations of Acalles parvulus, but not a single specimen of Acalles temperei has ever been collected in this area: [Fig. W33.4]. 

Since 1997 one of the authors (P.E. Stüben) visited many of the collecting localities of both species in central, western and southern Europe. Today the European distribution and its limits as well as the abundance of populations is well known for both taxa due to intensive collecting. Altogether, P.E. Stüben collected 708 specimens in 137 localities. The material has been predominantly obtained by shifting from leaf litter in deciduous forests. The preparation always included examination of the aedeagus. All material belongs to the collection of P.E. Stüben. In addition, 739 specimen from 259 further localities from several European museums and from colleagues' collections have been examined and determined:

[DaParv][DaTEM].

The aedeagus-based differential diagnosis for the distinction of both species presented by Péricart is convincing, easily comprehensible and sufficient for a quick determination of the material: [Fig. W33.5][Fig. W33.6]. 

2.2 Reconstruction of a cline in the Départements Loire and Isère (France) 

One of the authors (P.E. Stüben) was surprised by a phenomenon - unexpected in Cryptorhynchinae to such extent - that he discovered for the first time while collecting in the Département Isère (France). In the vicinity of the villages Saint-Jean-de-Bournay and Côte-Saint-André, about 40 km southeast of Lyon, he collected specimens of Acalles on several hills in an altitude between 200 and 600 meters above sea level. It proved impossible to unambiguously determine these specimens based on the form of their aedeagi. P.E. Stüben extended the investigation area during December 1999 and in the years 2005 and 2006 eastwards to the surroundings north of Grenoble (Mont-Saint Martin) and westwards from the western side of the Rhone valley up to the Parc Regional Mont Pilat - an area he had already visited in 1997. In the first instance the variability of different shapes of the aedeagus did not allow any conclusive interpretation: [Fig. W33.7]. From an acute-angled-inclined to an acute-angled-rounded and an obtuse-angled-rounded to an obtuse-angled-inclined median lobe, there was often a confusing mixture of aedeagus shapes represented together on the same hill. 

Are there different parapatric varieties of a single species, interbreeding along a narrow borderline? Stringing the aedeagus shapes along a transect between Mont Pilat near the village Pélussin (Dép. Loire) and Mont Saint-Martin north of Grenoble (Dep. Isère) a cline becomes apparent. [Fig. W33.8] This cline starts in the western populations with a rounded aedeagus 'typical' for Acalles parvulus and 'condenses' through a smooth transition in the eastern populations on the hills near Saint-Jean-de-Bournay and Côte-Saint-André to an aedeagus shape that doubtlessly and without any exception has to be ascribed to Acalles temperei. 

Is it possible for such a cline to develop between formerly isolated populations (under different selective pressures), allowing gene flow between them subsequent to a glacial isolation? One could imagine such a scenario only if the genetic divergence between the populations did not become too marked during this (glacial?) period. Wägele underlines that the (phylogenetic) species concept requires definitive evidence for the irreversible genetic divergence of populations [Wägele 2001: 58]. Considering the geographic pattern of variation along the mentioned cline, the question whether there are two species or one seems to be answered 'morphologically'. This would lead the taxonomist to consider Acalles temperei Péricart, 1987 a junior synonym of Acalles parvulus Boheman, 1837!

But what about the genetic divergence of these morphologically varying populations?

2.3 Molecular analysis of genetic distances 

As part of an integrative taxonomy project combining morphology and DNA taxonomy, one of the authors (J.J. Astrin) molecularly analyzed 18 species of Cryptorhynchinae based on 52 specimens. Collecting localities were clustered in two regions, separated from each other by a distance of about 600 km [Material].

DNA sequence data were collected from September 2005 until January 2006 in the molecular laboratory of the Zoological Research Museum Alexander Koenig (Germany: Bonn).

Three genes (subject to different evolutionary mechanisms or stemming from different genomes within the animals) were sequenced to obtain genetic distance values (p-distance): the protein-coding cytochrome c oxidase subunit1 (CO1) gene and the 16S rRNA (LSU) gene, both of mitochondrial origin, along with the nuclear 28S (LSU) ribosomal RNA gene. Fragment lengths were:

• CO1: 653 base pairs (bp) of the 5' region
• 16S: 411 bp of the 3' region
• 28S: two indel regions (i.e. gene regions affected by insertion/deletion events) with 201 bp (aligned) from a 548 bp fragment of the 3' region.

Primer and amplification data as well as DNA sequences will be published elsewhere.  

CO1 is the preferred marker for "DNA barcoding" studies [Hebert et al. 2003]. The suitability of 16S [Schubart et al. 2000][Vences et al. 2005][Astrin et al., in press] or of 28S [Markmann & Tautz 2005] has also been highlighted based on the favorable taxonomic signal or on certain molecular properties. 

Each of the three markers could (re-)identify the sampled Cryptorhynchinae according to morphological hypotheses in a fast tree-based approach (neighbor joining), as the species always grouped in a reciprocally monophyletic way (with the exception of Acalles temperei and Acalles parvulus, see below). Collectively comparing intraspecific and interspecific distances over the whole dataset, variation was clearly discontinuous between them, forming two distinct groups. This can be visualized particularly well through a box plot (on box plots in taxonomy, cf. [Astrin et al., in press]):

[Fig. W33.9].

Hints highlighting evolutionary interesting cases and/or species of dubious taxonomy can be gained thanks to this obvious 'taxonomic gap' between genetic distance values.  

Thus, when considering the box plots [Fig. W33.9], it can be noticed that the distances for the pair Acalles temperei / Acalles parvulus fall within the intraspecific range. In the mitochondrial tree reconstructions, a reciprocally monophyletic sub-structuring recovering Acalles temperei and Acalles parvulus became apparent (albeit based on very short branches). However, the nuclear marker does not recover such a topology: for 28S, one of the individuals of Acalles parvulus collected in the Isère district (see above) grouped with the Acalles temperei samples from the same location. This suggests the existence of hybridization events between both groups. 

Preliminary conclusion: So far, 5 individuals of Acalles parvulus and 2 of Acalles temperei have been analyzed. For definitive conclusions, more animals (preserved in absolute ethyl alcohol) would be necessary, particularly from the locus typicus in the Pyrénées-Orientales. However, the results obtained here already give evidence for Acalles parvulus and Acalles temperei being a single species that probably presents geographic structuring. 

3. Kyklioacalles roboris (Curtis, 1834) and
Kyklioacalles navieresi (Boheman, 1837) 

3.1 Morphology, biogeography and ecology 

In 2003 one of the authors (P.E. Stüben) resynonymized and redescribed the species Acalles navieresi Boheman, 1837 and, together with Acalles roboris Curtis, 1834, transferred it to the genus Kyklioacalles Stüben,1999 in his "Revision of the genus Kyklioacalles and description of the subgenus Palaeoacalles subg. n. in consideration of phylogenetical, morphological und biogeographical aspects". [Stüben 2003a: 116-166]

With only a few exceptions (the European High North and northern Africa), the new subgenus Palaeoacalles spreads over the whole western Palearctic region. In Central Europe, its 'core area', it is represented by Kyklioacalles roboris [Fig. W33.11], Kyklioacalles navieresi [Fig. W33.10] and Kyklioacalles velebitensis Stüben 2005 [Fig. W33.12] (the latter described from Croatia in 2005). A comprehensive differential diagnosis exists for these three species. [Stüben et al. 2005: 102] 

Based on this differential diagnosis, on observations regarding the host plant and substratum as well as on the palearctic and parapatric distribution of Kyklioacalles roboris and Kyklioacalles navieresi [Fig. W33.13], one of the authors (P.E. Stüben) presented the distribution in the Rhineland in detail [Fig. W33.14], particularly with regard to the common occurrence of both species on the Bausenberg (Germany: Eifel), and discussed the different ecological requirements. [Fig. W33.15] Thus the thesis that for most of Europe, Kyklioacalles roboris is the more hygrophilic (and darker) species has to be supplemented by the fact that Kyklioacalles navieresi is definitely the more xerothermic (and paler) species. [Stüben 2005a] 

Only thereby (ecological difference), selective collecting of Kyklioacalles navieresi became possible - for instance on chalky soils or on volcanic cinder soils of extremely xerothermic slopes [Fig. W33.16]. Nevertheless, and regardless of the straightforward determination of both species based on the shape of the aedeagus [Fig. W33.17] and the structure of the internal sack [Fig. W33.18][Fig. W33.19] (morphological difference), there still remains some doubt for the unexperienced observer trying to distinguish between the two species and using only ectoskeletal characters. The successful determination of females depends on the comprehensive consideration of all ectoskeletal characters (in detail: [Stüben 2003a / 2005a]). However the difficulties which are associated with determination raise new doubts regarding the species status of Kyklioacalles navieresi. It should be clear that this is mainly a problem of scientific communication and particularly a problem of macrophotographic illustration, to which ongoing work already pays attention. [Fig. W33.20] 

Doubts regarding the species status of Kyklioacalles navieresi could not be resolved by reference to macrophotography. However this issue may be comparable to the problem concerning Acalles parvulus-temperei discussed above. Here as well, the two species of Kyklioacalles are hard to distinguish using ectoskeletal characters. Females are often left in the collection without being determined. [Fig. W33.21] The two species are parapatric (Eifel), and in very rare cases, specimens of both species can be collected in the same sample by shifting at different places over a wide area. 

Are we again confronted with two different, parapatric varieties of the same species which interbreed along a narrow borderline? Until the end of 2005, one of the authors (P.E. Stüben) collected and examined 1644 specimens from 247 localities in an area which corresponds approximately to the distribution range of Acalles parvulus. Today, most of the specimens belong to the collection of P.E. Stüben:

[DaROB][DaNAV].

But there were neither differences nor even slight variations concerning the shape of the aedeagus or the internal sack, nor a cline as described above for Acalles parvulus-temperei! 

This is the more striking because the areas of distribution of both Kyklioacalles species overlap and interpenetrate [Fig. W33.13][Fig. W33.14] much more often than the distinctly delimited areas of distribution of both Acalles parvulus 'varieties' [Fig. W33.4]. If, according to Mayr, species are defined as groups of actually or potentially interbreeding populations [Mayr 1942], it has to be noted that the former is certainly not the case in the Kyklioacalles species here considered. Despite their 'spacial closeness', they live reproductively isolated (there exist other ecological parameters). In light of this morphological and ecological divergence one may suppose that they passed through a speciation process (not necessarily allopatric, e. g. due to a glacial separation). This speciation process would imply an irreversible divergence of the earlier populations.

However nobody would share the idea of accepting only the morphological and ecological similarity as species classification criteria. And surprising constellations as in the case of the detection of the Acalles parvulus-temperei-cline can never be entirely excluded. Without crossbreeding experiments which would conclusively determine the issue (if one is not willing to abandon the "biological species concept"!), the genetic distance remains as another useful evidence.

What about the genetic divergence of the Kyklioacalles species? 

3.2 Molecular analysis of genetic distances 

For DNA sequence analysis, two specimens per collecting region and per species were used (total 8 specimens). In contrast to the pairwise comparisons of Acalles temperei and Acalles parvulus, the distances between Kyklioacalles navieresi and Kyklioacalles roboris lie within the usual congeneric range (CO1, 28S) or between the within-species and between-species 'blocks' (16S):

[Fig. W33.9].

The interspecific distances for Kyklioacalles navieresi - Kyklioacalles roboris and for Acalles temperei - Acalles parvulus are given below.

The Kyklioacalles species pair reached values from almost twice to almost five times as high as those for Acalles temperei - Acalles parvulus. Note that the Kyklioacalles roboris intraspecific distances partly had values as high as those assumed to be between-species distances for Acalles temperei and Acalles parvulus. [Fig. W33.9] 

Conclusion: Independently from the comparison to Acalles temperei - Acalles parvulus, based on these first molecular data it can be said that Kyklioacalles navieresi and Kyklioacalles roboris likely represent two individual, but closely related species. 

Acknowledgment 

W. Wägele (Zoological Research Museum Alexander Koenig) kindly provided the materials required for molecular work in the pilot project on integrative taxonomy in Cryptorhynchinae.  

References 

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Markmann, M. & Tautz, D. (2005): Reverse taxonomy: an approach towards determining the diversity of meiobenthic organisms based on ribosomal RNA signature sequences. Philosophical Transactions of the Royal Society of London Series B - Biological Sciences 360, 1917-1924.
Mayr, E. (1942): Systematics and the origin of species. 334 pp., New York: Columbia University Press.
Péricart, J. (1987): Une espèce nouvelle d’Acalles des Pyrénées-Orientales: Acalles temperei, n. sp. (Coleoptera, Curculionidae). - L’Entomologiste, Paris 43(4): 193-196.
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Addresses of the authors:
Dr. Peter E. Stüben 
CURCULIO-Institute 
Hauweg 62, D- 41066 Mönchengladbach, Germany 
E-Mail: P.Stueben@t-online.de

Jonas J. Astrin 
Zoologisches Forschungsmuseum A. Koenig 
Adenauerallee 160, D-53113 Bonn, Germany 
E-mail: J.Astrin.ZFMK@uni-bonn.de