Introduction

The Western Palearctic genus Aporophyla Guenée, 1841, with a Ponto-Mediterranean distribution, occurs from the Atlantic coasts in the west, to eastern Turkey, Iraq and western Iran in the east. Six species of this genus are known from Europe, three of which - Aporophyla lueneburgensis (Freyer, 1848), Aporophyla lutulenta ([Denis & Schiffermüller], 1775) and Aporophyla nigra (Haworth, 1809) - occur in central Europe ( Ronkay et al. 2001).

The taxonomic separateness of A. lueneburgensis as a species has been debated by lepidopterologists since the late 19 ^th century. Some authors were inclined to accept the specific separateness of this taxon. Ultimately, however, it came to be regarded as a synonym of A. lutulenta ( Buszko & Nowacki, 2000; Nowacki, 1998; Nowacki & Fibiger, 1996). This view prevailed because of the great external similarity of the two species, despite the considerable individual variation within each group, especially as the type localities in Europe, given in the original descriptions of both taxa, are not so far apart: the Vienna region for A. lutulenta and Lüneburg (Lower Saxony) in Germany in the case of A. lueneburgensis ( Orhant, 2012). However, meticulous examination of the structure and functioning of the genitalia of both species were crucial for restoring the specific status of A. lueneburgensis ( Ronkay et al. 2001). At the same time, the ranges of the two species in Europe are generally allopatric. A. lueneburgensis is an Atlantic-Mediterranean species distributed from Spain and Portugal, through northern Italy, Switzerland, France, Great Britain, the Netherlands, Belgium, Denmark, Germany and southern Sweden, reaching its eastern boundary in Austria, the Czech Republic, Poland, Lithuania, Latvia, and southern Finland. In contrast, its sister species A. lutulenta has a Pontic distribution in south-eastern Europe, from the Caspian Sea across the steppes of southern Russia, Ukraine, all the countries on the Balkan Peninsula and Italy as far as central Europe, where it has been recorded in Hungary, the Czech Republic, Slovakia, Austria, south-eastern Germany and southern Poland ( Aarvik et al. 2017; Buszko & Nowacki, 2017; Rákosy, 1996; Ronkay et al. 2001; Varga et al. 2005). It must be emphasized that the distributions of A. lutulenta and A. lueneburgensis to the north of the Carpathians and Sudetes are distinctly allopatric, and their range boundaries meet in Poland. Hitherto, A. lutulenta was thought to inhabit south-eastern Poland; its localities in southern Podlasie and Polesie clearly lie on its north-western range boundary. In contrast, A. lueneburgensis was earlier reported solely from western and central Poland, at numerous localities from Pomerania, through central Wielkopolska, as far as Lower Silesia ( Buszko & Nowacki, 2017). In the light of recent records of A. lueneburgensis from Lithuania, Latvia and southern Finland ( Aarvik et al. 2017), it appears crucial to re-examine the specific status of specimens regarded as A. lutulenta, reported earlier from eastern Poland ( Nowacki, 2002).

Using genetic techniques, Orhant (2012) attempted to resolve the doubts surrounding the specific separateness of A. lueneburgensis and A. lutulenta from western Europe. However, his erroneous interpretation of the results led him to infer that only A. lutulenta was present in Europe, and that A. lueneburgensis was its junior synonym. In contrast, Andrillo (2019) expressed a different opinion. Based on the genetic code of the remains of A. lueneburgensis discovered in bat guano, he was able to document the presence of that species in the Geneva region of Switzerland, at the same time accepting that A. lutulenta was an eastern European species.

In view of the above, we set up the hypothesis that the two sister species A. lueneburgensis and A. lutulenta, occurring allopatrically in Poland, though exhibiting only slight morphological differences, are genetically different. This may be due to the historical breakup of the contiguous distribution of a precursor species in Europe and the subsequent long-term isolation of its geographical populations.

For these reasons, the aim of this research was to perform a morphological analysis and also an analysis of genetic separateness based on molecular studies of specimens from isolated populations from western Poland regarded as A. lueneburgensis and from eastern Polish populations regarded as A. lutulenta.

Materials and methods

The research material, in the form of imagines of both species, was accumulated from 2005 until 2019 during fieldwork at the following localities: for A. lueneburgensis in western Poland at Biedrusko, Bielinek, Borne Sulinowo, Marianka ad Brody, Poznań and Strzeszyn, and for A. lutulenta in eastern Poland at localities in five regions: Podlasie - Mielnik; Polesie - Macoszyn, Kosyń, Wola Uhruska; Bieszczady Mts. - Krzywe; Beskid Mts. - Umieszcz; and Pieniny Mts. - Sromowce Niżne. The moths were caught at night, having been attracted to a white screen illuminated by a 250 W mercury vapour lamp. Each specimen intended for the molecular studies was preserved in ethanol.

- A. lueneburgensis: western Poland: Bielinek 23-IX-2016, 1 ex., leg. R. Wąsala, Borne Sulinowo 26-V-2016 ex-larva, 1 ex., leg. A. Berezowski, Marianka ad Brody 18-IX-2012, 1 ex., leg. R. Wąsala, Poznań 2-X-2016, 2 exx., leg. R. Wąsala, Strzeszyn 25-IX-2010, 1 ex., leg. Ł. Matuszewski.

- A. lutulenta: eastern Poland: Mielnik 17-IX-2015, 1 ex., leg. D. Wasiluk, Macoszyn 16-IX-2015, 1 ex., leg. M. Hołowiński, Kosyń 7-IX-2019, 1 ex., leg. M. Hołowiński, Wola Uhruska 30-IX-2016, 2 exx., leg. Ł. Dawidowicz, southern Poland: Krzywe 10-IX-2013, 2 exx., leg. Ł. Matuszewski, Umieszcz 15-IX-2014, 1 ex., leg. K. Mazur, and Sromowce Niżne 31-VIII-2005, 1 ex., leg. R. Wąsala.

The individuals intended for morphological analysis were killed with ethyl acetate, then mounted in the usual way for lepidopterans. Preparations of male and female genitalia were also made.

Morphological analysis

The span, structure and coloration of both pairs of wings were compared in 10 individuals of each species. The genitalia of males and females were also compared: 5 preparations each of males and females of both species were made in accordance with the technique given in Nowacki (1995).

Molecular analysis

For the molecular analysis two legs were taken from 10 individuals of A. lutulenta and from 10 of A. lueneburgensis. DNA was extracted using Macherey-Nagel’s Nucleospin Tissue extraction kit, following the manufacturer’s protocol. Amplification of mitochondrial gene COI was performed in a volume of 20 μl, in one or two parts, depending on the sample condition. The PCR protocol followed Wahlberg and Wheat (2008) with the following primer pairs (Simón et al. 1994; Folmer et al. 1994): LCO-1940 / HCO-2198 for COI sequenced in one piece; LCO-1940 / K699 and C1-J-1751(alias Ron) / C1-N-2191(alias Nancy) for COI sequenced in two pieces. The PCR products were sent for purification and sequencing to Macrogen Europe (Amsterdam, Netherlands); all the other analyses were conducted in the molecular laboratory of the Nature Education Centre of the Jagiellonian University, Kraków.

An additional 24 sequences of four species of Aporophyla were imported from the GenBank and BOLD Systems databases: A. lutulenta, A. lueneburgensis, A. nigra and A. australis (Boisduval, 1829). The final matrix involved 41 nucleotide sequences with a total of 612 positions in the final dataset. All sequences were aligned manually in Bioedit, version 7.0.9.0. ( Hall, 1999). Evolutionary analyses were conducted in MEGA X ( Kumar et al. 2018). Pairwise distances were calculated using the Maximum Composite Likelihood model ( Tamura et al. 2004) and the pairwise deletion option. A Maximum Likelihood tree was inferred using the Tamura 3-parameter ( Tamura, 1992) model and the partial deletion option. The branch support for internal nodes was measured using 1000 rapid bootstrap replicates. The final tree was edited in Corel DRAW 2018 to enhance picture quality. The analyses were performed in the Molecular Laboratory of the Nature Education Centre of the Jagiellonian University.

Estimation of divergence times

Divergence times were estimated using the COI gene in Beast V.2.6.3 ( Bouckaert et al, 2019) using the StarBEAST2 package ( Ogilvie et al. 2017). The GTR nucleotide substitution model with four gamma categories and estimated base frequencies was implemented, and the three codon positions were unlinked in order to be estimated independently. For this study, the constrained root age was obtained by using the results of Wang et al. (2014) as a secondary calibration, so that three secondary points were used: 1 ^st: Egira acronyctoides Wileman, 1914, Pseudopanolis heterogyna Bang-Haas 1927, Panolis flammea ([Denis & Schiffermüller], 1775), Panolis pinicortex Draudt, 1950 (mean: 8.17), 2 ^nd: P. flammea, P. heterogyna and P. pinicortex (mean: 7.31, 95% HPD: 4.99-9.87), and 3 ^rd: P. flammea and P. pinicortex (mean: 6.54, 95% HPD: 4.52-8.87). Using the strict molecular clock model, the conventional mutation rate was fixed for the arthropod mitochondrial COI gene from the literature: 2.3% (0.0115 substitutions/site/million years) (Browner 1994; Wang et al. 2014; Nowacki et al. 2019). A Yule speciation process model was selected and two independent MCMC analyses were run for 30 000 000 generations, with Markov chains sampled every 5 000 generations. The results of the two runs were combined in LogCombiner 2.6.3 with the initial 10% of the trees discarded as burn-in. Tracer V. 1.7 ( Rambaut et al. 2018) was used to determine convergence, measure the effective sample size of each parameter, and calculate the mean and 95% highest posterior density (HPD) intervals for the divergence times.

Results

The analyses confirmed the occurrence in Poland of both sister species A. lueneburgensis and A. lutulenta. Importantly, however, genetic analysis provided incontrovertible evidence that all the moths from Polesie and Podlasie in eastern Poland belonged to A. lueneburgensis. This will therefore require a revision of the earlier information given by Nowacki (2002) regarding the distribution of A. lutulenta in this region of Poland. By contrast, the occurrence of A. lutulenta in southern Poland was confirmed, i. e. in the Bieszczady, Beskid and Pieniny mountain ranges of the Carpathians.

Morphology

Morphological analysis of the two species revealed only slight differences between them. A. lueneburgensis from Poland has slightly narrower, clearly pointed forewings. The coloration is variable, from ash grey through brown to brownish-black, and the inner, outer and wavy cross-lines (bands) are clearly visible. The kidney-mark and oval are also fairly distinct ( Figure 1). The wingspan is 33-38 mm. A. lutulenta from southern Poland has slightly broader forewings with slightly more rounded tips. In coloration it is more uniformly tawny to brown, the cross-lines are faint and the kidney-mark and oval are almost invisible ( Figure 2). The wingspan is 35-40 mm.

Analysis of the genitalia in several individuals of both species did not reveal any substantial differences in their overall structure, either in males or in females. There are, however, some conspicuous differences in the organs of both sexes. In males the shape of the vesica everted from the aedeagus is different. In A. lutulenta, a section of the vesica corresponding to half the length of the aedeagus is only slightly deflected from the longitudinal axis of the aedeagus; beyond that section, there is another slight flexure of the vesica. The shape of the everted vesica in A. lueneburgensis is quite different: immediately beyond the aedeagus it is strongly deflected from the former’s longitudinal axis. Also, the cornuti present at the base of the vesica are distinctly different in the two species ( Figure 3). In the case of the females of both species a slight difference is perceptible in the structure of the ostium bursae: in A. lutulenta it is shorter and connected with the ductus bursae by a short membranous section, while in A. lueneburgensis it is longer and connected with the ductus bursae by a longer membranous section. There are particularly conspicuous differences in the structure of the ductus bursae. In A. lutulenta this is longer and for most of its length is membranous with a few stronger ribs, and only the proximal and distal ends are strongly sclerotized. In A. lueneburgensis, however, the ductus bursae is shorter and strongly sclerotized with just a short membranous section in the middle part. A slight difference is also discernible in the appendix bursae. In A. lutulenta it is clearly shaped and takes the form of an extended oval bulge, from the middle part of which the ductus seminalis emerges. By contrast, the appendix bursae in A. lueneburgensis is slightly smaller, taking the form of a short, less regular bulge, with the ductus seminalis emerging from its distal end ( Figure 4). Additionally, while it was not subject of analysis in this study, it is worth mentioning that differences between studied species are also noticeable in the adult caterpillars of said species (Wegner, 2021).

Genetic analysis

Genetic analysis of specimens of A. lutulenta and A. lueneburgensis from Poland showed unequivocally that all the moths (6 exx.) from the localities in western Poland, i. e. Bielinek, Borne Sulinowo, Marianka ad Brody, Poznań and Strzeszyn, belonged, as anticipated, to A. lueneburgensis. It turned out, moreover, that all the individuals from eastern Poland, i. e. Podlasie (1 ex.) and Polesie (4 exx.) from the localities at Mielnik, Macoszyn, Kosyń and Wola Uhruska, also belonged to A. lueneburgensis. In contrast, all the moths from southern Poland (4 exx.) from the localities at Krzywe (Bieszczady Mts.), Umieszcz (Beskid Mts.) and Sromowce Niżne (Pieniny Mts.) belonged to the sister species A. lutulenta.

The results of the phylogenetic analyses confirm that A. lutulenta and A. lueneburgensis belong to two distinct yet closely related species ( Figure 5). The mean genetic distance between the A. lutulenta and A. lueneburgensis species groups was 0.02, whereas the distance between any other two of the examined species’ groups was slightly greater, i. e. between 0.04 and 0.06. The mean within-group distance was the shortest in the lueneburgensis and the australis groups (0.002), longer in the nigra group (0.004) and the longest in the lutulenta group (0.008). The pairwise distances between all the specimens examined are shown in Table 1.

The inference from the StarBEAST2 analysis is that the genus Aporophyla diverged during the Pleistocene (mean: 2.69 Mya, 95% HPD: 1.51-3.72) ( Figure 6), which makes Aporophyla a relatively young genus within Noctuidae. Moreover, A. australis diverged from its sister species A. nigra 1.46 Mya (95% HPD: 0.3-2.32), and A. lueneburgensis from its sister species A. lutulenta 1.3 Mya (95% HPD: 0.5-2.12).

Discussion

The results of this research have confirmed that populations of the two sister species A. lueneburgensis and A. lutulenta in Poland are allopatric. It turned out, however, that the populations from eastern Poland (Podlasie and Polesie) belong not to A. lutulenta, as thought earlier ( Nowacki, 2002), but to A. lueneburgensis. In the context of the latest records of A. lueneburgensis from Latvia and Lithuania ( Aarvik et al. 2017), this result comes as no surprise. Morphological analysis of specimens of the two species has shown that they differ only slightly in shape, coloration and wingspan. But as there is considerable individual variation within each species, these morphological differences cannot be used for a reliable discrimination between them.

Similarly, analysis of the male and female genitalia revealed only slight differences in their overall structures. However, there were significant differences in the morphologies of the vesica everted from the aedeagus (male) and in the ostium bursae, ductus bursae and appendix bursae (female). These differences may effectively prevent spermatophore transfer to the female bursa copulatrix during the second stage of copulation. It seems, therefore, that the lock-and-key systems in the internal genitalia can prevent successful copulation between individuals of the two species ( Lafontaine & Mikkola, 1987; Mikkola, 1993).

Genetic analysis showed that individuals of A. lueneburgensis from western and eastern Poland differed distinctly from those of A. lutulenta obtained from the Bieszczady, Beskid and Pieniny mountain ranges in south-eastern Poland. The calculated genetic distance between individuals of these two sister species was 0.02, which confirms their taxonomic separateness. For comparison, the genetic distance calculated for pairs of the morphologically distinct species A. australis and A. nigra is slightly greater at 0.04. What is surprising is the relatively large difference between the mean intra-group distance in A. lueneburgensis and A. lutulenta. It was the smallest (0.002) among the specimens of A. lueneburgensis, which testifies to the minimal genetic differentiation of this species in Europe. This may be due to the distribution of A. lueneburgensis being contiguous in western Europe, where uplands and lowlands are dominant and there are no distinctive barriers separating particular populations. This stands in contrast to A. lutulenta, in which the mean intra-group distance was 0.008, the highest value of any of the four Aporophyla species. This indicates the considerable dispersion and isolation of A. lutulenta’s various populations in Europe, which is hardly surprising since it occurs in those parts of Europe dominated by mountains, from Italy and the eastern Alps and Carpathians to the south and east across the Balkan Peninsula and further as far as the Near East ( Figure 7).

This is confirmed by the results of Orhant’s (2012) comparison of a number of morphologically diverse specimens, superficially resembling both species, from western Europe, i. e. from Belgium and France to western Austria and Germany. He inferred from the results of his genetic study that all the specimens belonged to the same species, A. lutulenta, and that A. lueneburgensis was merely a synonym of the former. Orhant’s (2012) diagnosis was correct in the sense that he was dealing with one species, but crucially, the specimens he examined came from areas of Europe potentially inhabited exclusively by A. lueneburgensis.Andrillo (2019) expressed exactly the opposite opinion: he compared the genetic code of the remains of A. lueneburgensis discovered in bat guano from the Geneva region in Switzerland with sequences deposited in the BOLD gene bank and found that it did indeed indicate the presence of that species. On this basis he carried out a revision of earlier faunistic data, removing A. lutulenta from the list of Swiss Lepidoptera and replacing it with A. lueneburgensis. At the same time, he acknowledged that A. lutulenta was a species occurring in eastern Europe ( Andrillo, 2019). This hypothesis was supported by results relating to both species from Bavaria and Austria, where both species occur ( Haslberger & Segerer, 2016; Huemer et al. 2019). It turns out, then, that the high mountain ranges crossing central Europe, from the Alps to the Sudetes and western Carpathians, form the boundary between the distributions of A. lueneburgensis and A. lutulenta. In summary, those areas of Europe lying to the north of these mountain ranges, from the Iberian Peninsula to southern Scandinavia, can be assumed to be inhabited by A. lueneburgensis, while those to the south and east are home to A. lutulenta.

The results of this research confirm our hypothesis regarding the phylogeny and interrelationships of A. lueneburgensis and A. lutulenta. This proposed that as a consequence of the historical breakup of the contiguous population of the precursor species in Europe, populations of these moths became isolated from each other for an extremely long time, eventually evolving into two distinct, sister species. We can assume that the distribution of the precursor species was disrupted during the Pleistocene as a result of the Günz glaciation 1.2 - 0.95 Mya. During that period, northern and eastern Europe was covered by the ice-sheet whereas the remainder of the continent from the Pyrenees to the Black Sea was a periglacial zone with long-term permafrost ( Mojski, 1993). This hypothesis is confirmed by the approximate divergence time of A. lueneburgensis and A. lutulenta, calculated at c. 1.3 Mya. The precursor species’ original distribution having been split up, the isolated populations will have survived in two refuges: the Pyrenean one along the Atlantic coast and the Near Eastern one to the south of the Black Sea. The speciation of the two present-day sister taxa gradually took place during the subsequent hundreds of thousands of years of glaciation in Europe. Once the ice-sheet had begun to retreat from Europe, during the Holocene, the two new species gradually started spreading into the ice-free areas. A. lueneburgensis, being adapted to the Atlantic climate, moved mainly along the coasts of the Atlantic Ocean and North Sea, colonizing the whole of western Europe and progressively extending its range eastwards across Germany and into western Poland. In the late 20 ^th century, it expanded rapidly into the areas around the Baltic Sea: records from southern Sweden and Finland, as well as Lithuania and Latvia testify to this ( Aarvik et al. 2017). As our research has shown, it has also reached eastern Poland. A. lutulenta, on the other hand, evolved from that part of the precursor population isolated in the Near Eastern refuge and probably became adapted to the warmer Mediterranean climate. During the Holocene, it gradually spread westwards along Mediterranean coasts, eventually reaching the Alps and locally many sites to the south of the line formed by the Carpathians, Sudetes and Alps. This is a probable explanation for the allopatric distribution of the two species in Poland. It is certain that A. lutulenta occurs in southern parts of Poland, where it was recorded at Stuposiany (Bieszczady Mts.) ( Bieszczady, 1973) and also during our field studies. The earlier literature data pertaining to the occurrence of A. lutulenta in Western Pomerania ( Urbahn & Urbahn, 1939), Gdañsk ( Speiser, 1903) or Lower Silesia ( Wolf, 1935-1944) in fact relate to A. lueneburgensis, which has recently been recorded in eastern Poland as well.

Acknowledgments

We wish to thank Dr. Antonio Vives (Madrid, Spain) for translating the title and abstract into Spanish.

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Author notes

*Autor para correspondencia / Corresponding author