Molecular taxonomy of the Sympetrum vulgatum ( Odonata : Libellulidae ) complex in the West Palaearctic

The Sympetrum vulgatum (Linnaeus, 1758) complex is composed of the subspecies S. vulgatum vulgatum, S. vulgatum decoloratum (Selys, 1884) and S. vulgatum ibericum Ocharan, 1985 in the West Palaearctic. These taxa have parapatric distributions and noticeable morphological differences in colour and body size, and their taxonomic status is debated. Here we revise the systematics of this group using molecular taxonomy, including molecular analyses of mitochondrial (cytochrome c oxidase subunit I, COI) and nuclear (internal transcribed spacer, ITS1) DNA taking into account known morphological differences. Each subspecies has a unique and differentiated COI haplotype, although divergences among them are low (0.4% maximum uncorrected p-distance). The subspecies are not differentiated by the nuclear marker ITS1. The genetic results for these taxa contrast with the deep divergence of the sister species S. striolatum (Charpentier, 1840). Given current evidence, we propose to maintain the subspecifi c status of the S. vulgatum complex and hypothesize their biogeographical history. It is likely that the three subspecies became isolated during one of the latest glacial periods, each in a different refugium: S. vulgatum ibericum possibly occupied the Iberian Peninsula, S. vulgatum vulgatum the Balkan Peninsula or territories further east and S. vulgatum decoloratum Anatolia.

In addition, there are several species of Sympetrum that only differ in colouration or body size, which is a reason for doubting their validity.Molecular studies (Sawabe et al., 2004;Parkes et al., 2009) and others combining molecular and morphological analyses (Pilgrim & von Dohlen, 2007) conclude that in most cases there is no reason to maintain the specifi c status of some of them.However, there are still some cases that need clarifi cation (Boudot & Kalkman, 2015), including the status of the S. vulgatum subspecifi c complex.
Sympetrum vulgatum has a wide distribution in Eurasia, from Western Europe to the oriental part of Russia.It sometimes reaches as far as Britain in the west and Japan in the east (see distribution map in Boudot & Kalkman, 2015).Most of this huge distribution area is occupied by the nominotypical subspecies, but in the south it is replaced Molecular taxonomy of the Sympetrum vulgatum (Odonata: Libellulidae) complex in the West Palaearctic JOAN C. HINOJOSA1 , RICARD MARTÍN2 , XAVIER MAYNOU 2 and ROGER VILA 1

INTRODUCTION
Until the introduction of molecular systematics, the genus Sympetrum Newman, 1833 (Anisoptera: Libellulidae) included over 60 species divided into subgroups according to morphological criteria, especially the secondary genitalia of the male and the female vulvar scale.These groups are now considered artifi cial due to a lack of synapomorphies and the long-standing debate over which taxa should be included in the genus.Pilgrim & von Dohlen (2012), combining molecular and morphological methods, provided evidence for the monophyly of the genus in spite of the existence of some dubious taxa and proposed the use of Sympetrum sensu lato and sensu stricto.They divided the genus into six species-groups, but pointed out that the relationships between them were not satisfactorily settled.They conclude that the genus arose about 50 mya and that both dispersal and vicariance might have played an important role in its biogeographical history.Their preliminary estimates provide a divergence time for the species-groups of approximately 32-38 myr, possibly infl uenced by climate cooling and drying in the late Eocene and early Oligocene, leading to the fragmentation of populations.One of these groups is the vulgatum group, which has a Hol-water bodies with clayey bottoms and abundant vegetation like fl oodplains and the littoral zones of water reservoirs (Schröter, 2010) and in Turkey in headwater regions above the treeline (Ikemeyer & Schneider, 2014).
Several authors have highlighted differences in colouration and size of these subspecies (Ocharan, 1985;Dijkstra & Lewington, 2006;Grand et al., 2007), which indicate the need to clarify the taxonomic status of both taxa by means of molecular analyses (Grand et al., 2007;Dijkstra & Kalkman, 2012;Boudot & Kalkman, 2015).These differences are summarized in Table 1 and illustrated in Fig. 1.
The aim of this study is to clarify the phylogenetic relationships between the European and Near East subspecies: S. v. vulgatum, S. v. ibericum and S. v. decoloratum, based on mitochondrial and nuclear DNA markers in order to shed light on their biogeographical history and if needed to revise their taxonomic rank.

Specimens
Nuclear (internal transcribed spacer, ITS1) and/or mitochondrial (cytochrome c oxidase subunit I, COI) DNA fragments from a total of 30 individuals of S. vulgatum were sequenced for this study (Table 2).All specimens were preserved in 95% ethanol after collection, except those of S. v. decoloratum, which were dried in acetone, and stored at -20°C.These samples are deposited in the DNA and Tissues Collection of the Institute of Evolutionary Biology (IBE), Barcelona, Spain.Three COI and three by paler forms (Dumont, 1977;Ocharan, 1985).Thus, on the Iberian Peninsula and a minute part of southern France S. vulgatum ibericum Ocharan, 1985, and from Turkey to central Asia S. vulgatum decoloratum (Selys, 1884) occur (Boudot & Kalkman, 2015).In addition, there are two subspecies in East Asia: S. vulgatum fuscopterum (Belyshev, 1971) and S. vulgatum imitans (Selys, 1886).
Sympetrum vulgatum ibericum is restricted to thirty UTM 10 × 10 squares on the northern Iberian Peninsula, specifi cally in the eastern part of the Iberian System mountain range, the Pyrenees and Pre-Pyrenees and the northern sub-plateau (see distribution map in Díaz Martínez & Evangelio Pinach, 2015).Several formerly published records originated from confusion with Sympetrum fonscolombii and S. sinaiticum (Díaz Martínez & Evangelio Pinach, 2015).It is also present in one square in Andorra (Grand, 2004) and in three in the Pyrénées-Orientales department in France (Grand et al., 2007).Its distribution does not seem to overlap that of S. vulgatum vulgatum.Its habitat preferences are sunny stagnant waters such as marshlands, mountain lakes and gravel pits.It is believed to be signifi cantly threatened (Boudot & Kalkman, 2015).
The distribution areas of S. vulgatum decoloratum and the nominotypical subspecies seem rather intricate and diffi cult to delineate.They possibly overlap in Georgia and Central Asia and there are individuals showing intermediate colouration in Kyrgyzstan (Schröter, 2010).In these geographical areas it occurs in a variety of shallow  DNA extraction was done following the protocol described in Vodă et al. (2015).
LCO1490 and Nancy primers (GGTCAACAAATCATAAA-GATATTGG and CCCGGTAAAATTAAAATATAAACTTC, respectively) were employed for the COI amplifi cation, and 21 sequences of 676 bp were obtained.Conditions were: fi rst denatured at 92°C for 60 s, then 92°C for 15 s, 48°C for 45 s and 62°C for 150 s in 5 cycles and another 30 cycles in which the annealing temperature was 52°C with the fi nal extension step at 62°C for 7 min.
ITS1 was amplifi ed with 18S-forward and 5.8S-reverse primers (GATTACGTCCCTGCCCTTTG and CGATGATCAAGT-GTCCTGCA, respectively) under the following conditions: fi rst denatured at 94°C for 150 s, then 94°C for 30 s, 46°C for 60 s and 72°C for 60 s in 5 cycles and another 30 cycles in which the annealing temperature was 52°C with the fi nal extension step at 72°C for 10 min.As a result, 29 sequences were obtained and the longest fragments obtained were 481 bp.
PCR products were purifi ed and Sanger sequenced by Macrogen Inc. Europe (Amsterdam, the Netherlands).All sequences have been deposited in GenBank (accession nos in Table 2).

RESULTS AND DISCUSSION
The phylogenetic tree based on the mitochondrial COI marker (Fig. 2) recovered all Sympetrum vulgatum sequences in a highly supported clade.Within this clade, the subspecies S. vulgatum ibericum and S. vulgatum decoloratum were recovered as monophyletic (albeit with low support), and each taxon displayed a single haplotype, as also refl ected in the COI haplotype network (Fig. 3).Sympetrum vulgatum ibericum specimens were differentiated from the rest by two diagnostic nucleotide changes, and S. vulgatum decoloratum displayed a single private mutation.Thus the maximum genetic distance was that of Sympetrum vulgatum ibericum with respect to S. vulgatum decoloratum (3 mutations, 0.4% uncorrected p-distance), much lower than the typical divergences between generally accepted Odonata sister or closely related species (Rach et al., 2007;Damm et al., 2010;Froufe et al., 2013;Ferreira et al., 2016) and in concordance with documented intraspecifi c variation (Rach et al., 2007;Damm et al., 2010).In fact, genetic distances among the three taxa studied were minimal compared to that of the sister species S. striolatum, for which the minimum interspecifi c distance was 10.6%.
In the phylogeny based on ITS1 sequences (Fig. 4) S. vulgatum formed a highly supported clade, but monophyly was not retrieved for any subspecies, indicating a close evolutionary relationship among all the individuals of this species.In fact, there were only two sequences with one variant each that did not correlate with the subspecies.Sympetrum striolatum sequences were once again highly divergent (6.3%) from those of S. vulgatum.
There is a remarkable divergence of more than 10% in COI of S. vulgatum and S. striolatum.Based on the data available S. vulgatum ibericum and S. vulgatum decoloratum cannot be treated as different species and it is proposed that the current subspecies status should be maintained.There are morphological differences and they could indeed be compatible with both species and subspecies status, but the low genetic differentiation detected, especially when compared to that of the sister species, suggests a very recent origin for these populations.It is suggested that light-  coloured insects may be favoured in warmer climates for thermoregulatory reasons (Zeuss et al., 2014;Pinkert et al., 2016) and this might be one plausible explanation for the pale colour of the meridional S. vulgatum subspecies.The results presented indicate that it is likely these populations diverged in the Upper Pleistocene.We hypothesize that they became isolated during one of the latest glacial periods in different refugia, S. vulgatum ibericum on the Iberian Peninsula, S. vulgatum vulgatum on the Balkan Peninsula or further east, and S. vulgatum decoloratum possibly in Anatolia.In the current interglacial, their ranges expanded to the present-day generally parapatric distributions.It is possible that the limited extent of secondary sympatry that is now observed is a result of a certain degree of hybrid depression in crosses between subspecies or to some degree of ecological specialization, but assessing this would require additional studies at the contact zones.
Overall, we found a low genetic differentiation among S. vulgatum putative subspecies in the mitochondrial marker COI and no divergence in the nuclear ITS1, with values below the estimated thresholds for reproductive isolation in Odonata (Sánchez-Guillén et al., 2014).These results sharply contrast with those obtained at the interspecifi c level, as there is a remarkable divergence of more than 10% in COI in the sister species S. v ulgatum and S. striolatum.Based on the data available S. vulgatum ibericum and S. vulgatum decoloratum cannot be treated as different species and we propose that the current subspecies status should be maintained.It is important to obtain new evidence that sheds light on debated taxonomic questions in order to understand the evolutionary history of taxa, as well as to properly prioritize conservation efforts.In this regard, obtaining and combining ecological, morphological and molecular data is key to eventually reaching a consensus and stability in the systematics of Palaearctic Odonata.

Fig. 3 .
Fig. 3. H aplotype network based on COI with the minimum uncorrected p-distances between the subspecies indicated.Every mutation is marked with a bar and the circle size is proportional to the number of samples represented.Sample locations are marked with dots coloured to correspond with the haplotype.The number of samples used for ITS1 and COI is indicated if there is more than one per locality.The map also shows the approximate distribution of the three subspecies: S. v. ibericum in blue, S. v. vulgatum in red and S. v. decoloratum in yellow, based on Boudot & Kalkman (2015) and Díaz Martínez & Evangelio Pinach (2015).

Fig. 2 .
Fig. 2. COI gene tree obtained using Bayesian inference.Maximum likelihood bootstrap values and Bayesian posterior probabilities are indicated in this order for the key nodes.Scale units are presented in substitutions per site.

Fig. 4 .
Fig. 4. ITS1 gene tree obtained using Bayesian inference.Maximum likelihood bootstrap values and Bayesian posterior probabilities are indicated in this order.Scale units are presented in substitutions per site.
Pilgrim & von Dohlen, 2012 from two S. striolatum, the sister species according toPilgrim & von Dohlen, 2012, and one individual of S. eroticum were retrieved from GenBank.COI and ITS1 sequencing

Table 2 .
Samples used in this study with the specimen codes, original localities and GenBank accession numbers.