NORTH-WESTERN JOURNAL OF ZOOLOGY 11 (2): 304-315 ©NwjZ, Oradea, Romania, 2015 Article No.: 151201 http://biozoojournals.ro/nwjz/index.html

Morphologies tells more than molecules in the case of the European widespread albimana (Fabricius, 1787) (Diptera, )

Edina TÖRÖK1,2,*, Levente-Péter KOLCSÁR1, Avar-Lehel DÉNES1 and Lujza KERESZTES1

1. Hungarian Department of Biology and Ecology, Babeş-Bolyai University, 400006 Cluj-Napoca, Clinicilor 5-7, Romania, [email protected], [email protected] 2. Department of Ecology, University of Szeged, Szeged, Közép fasor 52, Hungary * Corresponding author, E. Török, E-mail: [email protected]

Received: 18. September 2014 / Accepted: 30. March 2015 / Available online: 07. November 2015 / Printed: December 2015

Abstract. The natural biodiversity crisis opens new perspectives in the field of faunistic or taxonomic research. Dipterans are of major importance in almost all types of ecosystems. In the present integrative study a comparative morphological analysis and molecular tools (mtCOI sequences) were used to test taxonomic hypotheses in the case of the which presents highly divergent morphological structuring throughout its range. The morphologically divergent structures are clearly separated along an altitudinal gradient. The typical P. albimana were identified mostly at low altitudes in Europe (Luxembourg, Germany, France and Hungary, but also in Romania in Dobrogea and Bulgaria in Stara Planina), between 10-350 m (lowland-hilly area). In contrast, divergent morphological structures were identified from Romania and Bulgaria (the Carpathians and Rhodope Mountains), between 450 to 1450 m (hilly-mountainous area). In contrast the standard BOLD mtCOI sequences (658 bp) frequently used in molecular taxonomy did not reflect a similar pattern and suggested recent area dynamics of populations from different isolated glacial refugia in Europe. Based on deep morphological and significant ecological differences here, we describe a new species, Ptychoptera incognita sp. n., a dipteran that shares common ecosystems with some important glacial relict plants from the Carpathians and Balkan area.

Key words: genetic analysis, mtCOI, morphological variability, geometric morphometry, glacial relics.

Introduction whole range of selected taxa are even scarcer (Pfenninger & Schwenk 2007, Renaud et al. 2012, Species are recognized as basic units in almost all Ashfaq et al. 2014). More data is available in the biological studies, but detection and delineation of case of some groups like Culicidae (Beebee & species boundaries is still under intensive debate Rowe 1999, Vinogradova et al. 2007), Chaoboridae (Dayrat 2005, Hausdorf 2011). The communication (Brendonk & Spitze 2006, Brendonk et al. 2009), gap between “classical” and molecular biologists Chironomidae (Hofmann 1990, Brooks & Birks have culminated in the so-called “taxonomy cri- 2000, Michailova et al. 2008, Istonia et al. 2009, sis” and the need to redefine the concept of the Orendt et al. 2009), and much less in others, like species emerged, parallel with developing new Pediciidae (Ujvárosi et al. 2010), Syrphidae (Mi- methodologies that studies species boundaries lankov et al. 2005, 2008) and Thaumaleidae (Papp from multiple, complementary perspectives & Földvári 2007). Especially within the European (Padial et al. 2010). The “integrative taxonomy” fauna, well researched through centuries of taxo- approach has been efficiently introduced for some nomic investigation, the integrative approach may vertebrate (Kotlik & Berrebi 2002, Malhotra & reveal heretofore undetected new species (Skev- Thorpe 2004) and invertebrate taxa (Pauls et al. ington et al. 2007, Kehlmaier & Almeida 2014, 2010), but much less so for a number of neglected Ujvárosi & Bálint 2012). High dipeteran diversity or difficult-to-access groups, like dipterans with important areas of endemism were already (Renaud et al. 2012, Kehlmaier & Almeida 2014, identified in Mediterranean regions (Jong 1998, Ujvárosi & Bálint 2012). Jong et al. 2008), but recent phylogenetic works Dipterans represent one of the largest radia- also suggest important cryptic diversity in the tions of organisms, making up about 15% of ani- Carpathians, Alps and in the Hungarian Middle mal species, but they are consequently neglected Ranges as well (Papp & Földvari 2007, Brendonk in taxonomic or ecology studies and most knowl- et al. 2009, Ujvárosi et al. 2009, 2010). edge of dipteran diversity is based mainly on re- The dipteran family Ptychopteridae comprises gional faunistic and chorological data (Yeates & only 80 recent species worldwide that are classi- Wiegmann 2005). Integrative studies covering the fied in two subfamilies, Ptychopterinae where Morphologies tells more than molecules in the case of the European widespread 305 only one genus Ptychoptera (70) belongs, and Bitta- adopted. In addition, specific male genital struc- comorphinae with other two genera, tures and wing pattern can be observed (2 species) and Bittacomorphella (8 species) (Roz- (Krzemiński, 1986). This species is widespread košný 1997, Wagner et al. 2007, Nakamura & Sai- from central Scandinavia to Italy and Spain and gusa 2009, Nakamura 2012, Paramonov 2012). from Great Britain to Russia and Turkey. How- Based on a small number of extant species some ever, in most regions the species is rather sporadic, authors suggest a relic status of the family (Zitek- having only a few collection data. Zwyrtek 1971, Rozkošný 1997, Bertone et al. 2008, P. albimana was considered monotypic and Lukashevich 2012). The family is apparently ab- there were no detailed data on morphological and sent from Australasian and Oceanic regions. In the genetic variability of the species in its range. Quite Palaearctic, 30 species were recorded (Rozkošný recently an important morphological difference 1997) so far. Fifteen Ptychoptera species that belong was noticed between different populations in to subgenera Ptychoptera Meigen, 1803 and Parap- Europe (Ujvárosi et al. 2011). In the present study tychoptera Tonnoir, 1919 are present in Europe we analyze the morphological and genetic vari- (Zwick & Starý 2003, Paramonov 2013). The two ability in allopatric populations of the species and subgenera are separated by the presence of a com- test taxonomic hypotheses using an integrative plex auxiliary sexual organ on the male sternite 3 approach. (Zwick & Starý 2003). Selected species of Ptychop- tera can readily be distinguished by the structure of the male terminalia, but in some cases patterns Material and methods of wing venation and antenna design should bring Sampling and data collection additional information in taxonomic decisions A total of 101 male individuals were collected either by (Freeman 1950, Peus 1958, Tjeder 1968, Deliné- the authors using hand nets or by different other dip- Draskovits 1983, Krzemiński 1986, Zitek-Zwyrtek terologists (Peter Zwick or were obtained by loan from 1971, Krzemiński & Zwick 1993, Stubbs 1993, the Hungarian National History Museum, Budapest, Andersson 1997, Rozkošný 1997). In contrast, mo- Hungary (HNHM)). The specimens of P. albimana origi- nate from most parts of the known range of the species lecular tools are completely neglected in recent (Fig. 1, Table 1). Part of the freshly collected material was taxonomy studies, even an important cryptic di- stored in 96% ethanol, other part mounted dry and de- versity, frequently detected by molecular analyses posited in the Zoological Museum of Babeş-Bolyai Uni- in a series of other aquatic that might be versity in Cluj-Napoca, Romania. Material was identified expected in Ptychopteridae as well (Pauls et al. by the authors, with the exception of the material from 2006, Ujvárosi et al. 2010). Germany (identified by P. Zwick) and Hungary (identi- Ptychoptera albimana was first described as fied by Á. Deliné-Draskovits). Outgroup taxa included three species of Ptychopteridae: Tipula albimana by Fabricius in 1787 having type (Linnaeus, 1758) from Romania, Ptychoptera scutellaris locality near Kiel, northern Germany. The holo- Meigen, 1818 from Mongolia and type originally deposited in the Natural History (Fabricius, 1781) from USA. Museum from Copenhagen, Denmark, is no more The right wing (or left wing if right was missing or available (Thomas Pape, pers. com.). This me- damaged) was prepared by detaching it from the dium-sized Diptera resembles tipuloids with body length of about 8-11 mm and wing having a length of 9-12 mm. The species was collected in different ecosystems from clear rivers and pods to strongly polluted waters such as outlets from sewage treatment waters (Stubbs 1993). In contrast, in northern Europe the species was reported from small streams with clear and cold water, especially at muddy banks and in wet mud beside ponds and lakes, which frequently have a relic-like fauna (Andersson 1997). This species can be readily distinguished from all other members of the family by having the first

metatarsomere of the hind legs white colored. Figure 1. Map of collection sites of Ptychoptera albimana: Based on this character, the name “albimana” was black points: morphotype A, white points: morphotype B.

306 E. Török et al.

Table 1. All specimens are assigned with codes and collecting sites explanation of abbreviations: PA- Ptychoptera albi- mana (morphotype A), PI- Ptychoptera incognita sp. n. (morphotype B), BC- Bittacomorpha clavipes, PS- Ptychoptera scutellaris, PC- Ptychoptera contaminata, ZMUBB - Zoological Museum of Babeş-Bolyai University in Cluj-Napoca, RO, HNHM - Hungarian Natural History Museum Budapest, HU, CMNH - Carnegie Museum of Natural History, Pitts- burgh, Pennsylvania, USA, A- Ptychoptera albimana (morphotype A), B- Ptychoptera incognita sp. n. (morphotype B), O- outgroup.

Morpho- Mor- Genetic code Male/ Collec- Localites Eleva- Latitude Longitude metric pho- (BOLD) Female tion tion Code type (m) PA-DE01 A - 11 m ZMUBB Germany, Schlitz 265 m 50.663 N 9.573 E PA-DE03 A - 1 m ZMUBB Germany, Grebin 33 m 54.194 N 10.482 E PA-LU01 A EUTIP589 1 m ZMUBB Luxemburg, Grevenmacher 312 m 49.814 N 6.403 E PI-SR01 B? EUTIP588 1 f ZMUBB Serbia, Gostun 848 m 43.204 N 19.749 E PI-RO01 B EUTIP-584, 585 2 m ZMUBB Romania, Voşlăbeni 907 m 46.671 N 25.661 E PI-RO03 B EUTIP-590, 591, 15 m ZMUBB Romania, Cluj-Napoca 456m 46.721 N 23.528 E 593, 594, 596, 597, 598, 599 PA-RO02 A EUTIP583 1 m ZMUBB Romania, Luncăviţa 151 m 45.198 N 28.342 E PI-RO04 B - 12 m ZMUBB Romania, Poiana Mărului 1450 m 45.347 N 22.646 E PI-RO05 B - 1 m ZMUBB Romania, Feleacu 680 m 46.702 N 23.588 E PI-RO06 B - 1 m ZMUBB Romania, Anieş 720 m 47.463 N 24.762 E PI-BG01 B EUTIP587 1 m ZMUBB Bulgaria, Velingrad 870 m 41.964 N 23.944 E PA-BG02 A EUTIP-578, 579 1 m, 1 f ZMUBB Bulgaria, Dabravka 350 m 43.655 N 22.628 E PI-BG03 B EUTIP586 1 m ZMUBB Bulgaria, Velingrad 867 m 41.976 N 23.959 E PA-FR01 A EUTIP-580, 581, 3 m ZMUBB France, Lanslebourg Mont Cenis 275 m 45.367 N 5.996 E 582 PA-HU01 A - 18 m HNHM Hungary, Szilvásvárad 345 m 48.078 N 20.406 E PA-HU02 A - 4 m HNHM Hungary, Bakonyszentkirály 300 m 47.370 N 17.875 E PA-HU03 A - 1 m HNHM Hungary, Kemence 340 m 47.992 N 18.945 E PA-HU04 A EUTIP-571, 573, 7 m ZMUBB Hungary, Szilvásvárad 350m 48.078 N 20.406 E 574, 575, 576, 577 PA-HU05 A - 9 m HNHM Hungary, Zirc 390 m 47.260 N 17.878 E PA-HU06 A - 3 m HNHM Hungary, Csopak 186 m 46.969 N 17.920 E PA-HU07 A - 1 m HNHM Hungary, Bakonynána 300 m 47.270 N 17.991 E PA-HU08 A - 2 m HNHM Hungary, Szokolya 350 m 47.914 N 18.983 E PA-HU09 A - 1 m HNHM Hungary, Bakonyszentlászló 250 m 47.379 N 17.811 E PA-HU10 A - 1 m HNHM Hungary, Abaújlak 255 m 48.394 N 20.980 E PA-HU11 A - 1 m HNHM Hungary, Vilmány 144 m 48.424 N 21.206 E PA-HU12 A - 1 m HNHM Hungary, Diosjenő 300 m 47.956 N 19.044 E PA-HU13 A - 1 m HNHM Hungary, Kőszeg 300 m 47.359 N 16.509 E PA-UK01* A SATIP-952, 953 2 m CMNH United Kingdom, Aberdeenshire 13 m 57.172 N 2.103 W PA-PO01* A SATIP-856, 858 2 m CMNH Poland, Gdansk-Osowa 125 m 54.363 N 18.538 E PC-RO01* O EUTIP-600, 601 2 m ZMUBB Bulgaria, Dragoman 702 m 42.935 N 22.953 E PC-BG01* O EUTIP-602, 603 2 m ZMUBB Romania, Satu Mare 129 m 47.898 N 23.123 E PS-MO01* O SATIP-879, 880, 3 m CMNH Mongolia, Monkhkhayrkhan 1919 m 47.174 N 91.855 E 881 PS-MO02* O SATIP882 1 m CMNH Mongolia, Duut Soum 2270 m 47.533 N 91.640 E PS-MO03* O SATIP883 1 m CMNH Mongolia, Buyant Soum 2270 m 47.937 N 91.591 E BC-US01* O SATIP-894, 895 2 m CMNH United States 512 m 41.426 N 77.844 W BC-MO01* O SATIP-896, 897 2 m CMNH Mongolia, Arhangay 2230 m 47.757 N 99.358 E

original specimen and covering it with a glass plate prior Geometric and linear morphometry to the geometric measurements. For morphological Geometric morphometry was used to understand the measurements the abdomen of the male individual was variation in shape by removing non-shape variation ef- macerated in KOH 10% and then put in glycerol. Speci- fects independent of size (Bookstein 1997). For geometric mens were examined with an Olympus SZ61 stereomi- analysis 56 right and 31 left wings were fixed and photo- croscope equipped with Sony Alpha 200 camera. Species graphed. Position of 20 landmarks at the vein intersec- were identified using reference literature (Peus 1958, tions or terminations were designated and digitalized us- Krzemiński & Zwick 1993). ing TPSDig v2.16 (Fig. 2F). The coordinate data were im-

Morphologies tells more than molecules in the case of the European widespread 307

Figure 2. Measured characters on parts of male genitalia (A- E). A: dorsal view of 9th ter- gite and surstyle; B: ventral view of 9th tergite and surstyle; C: ventral view of style and tegmen; D: ventral view of aedoeagus; E: ventral view of hypandrium. F: Posi- tion of 20 landmarks and il- lustration of wing length (L) and width (W).

ported into MorphoJ v1.06a for shape statistical analysis. unique accession numbers were assigned in accordance First, Generalized Procrustean Analysis was used to re- with the Barcode of Life Data System (BOLD; move the non-shape variation which caused the effect of http://www.boldsystems.org), in a frame of the Ptychop- translation, rotation and the size of wings. A Principal teridae BOLD project. In molecular taxonomy analysis we Component Analysis (PCA) was constructed to test varia- used 658 bp length COI DNA barcodes. Sequences of the tion among individuals. Canonical Variate Analysis COI barcode region (Hebert et al. 2003) were generated at (CVA) was applied to determine relative differences the Canadian Centre for DNA Barcoding (CCDB), Uni- among two morphological groups (Bookstein 1997, versity of Guelph, Canada. Additional 13 Ptychopteridae Zelditch et al. 2004, Milankov et al. 2010, Klingenberg sequences were used in molecular analysis, which were 2011, Schutze et al. 2012). freely available in BOLD Database (sequences with SATIP Good landmarks on male genitalia structures were codes). The resulting Diptera barcodes were used to cor- hard to find; thus, linear morphometry was applied in the roborate the morphometric results with the molecular case of 101 male specimens. We quantified morphological data. variation among surveyed populations by comparing 19 The phylogenetic relationships within P. albimana morphological features of the male genitalia, which were inferred based on the whole dataset using a Maxi- showed high variability, making them suitable for our mum Likelihood (ML) and a Bayesian inference (BI) algo- studies (Fig. 2A-E). Discrete morphological characters rithm. Additionally, 13 sequences of 3 species were used were measured using ImageJ v4.46. The size (length and as outgroups in the phylogenetic analysis. jModeltest width) of the wing was also measured (Fig. 2F). Statistical (Guindon & Gascuel 2003, Darriba et al. 2012) was used to analyses were performed in R statistical environment test for the most suitable substitution model. Based on the v3.0.1. minimum value of the corrected Akaike Information Cri- teria, the General Time Reversible (GTR) model with Molecular methods and data analyses gamma-distributed variation rate across sites (G) and a For genomic DNA analysis we used legs of 30 identified proportion of invariable sites (I) was selected from a pool specimens (4 P. contaminata and 26 P. albimana), and of 88 models. The ML analysis was conducted in Seaview 308 E. Török et al. v4 (Gouy et al. 2010) using the PhyML option with the bimana, named below as morphotype A (the typi- starting tree determined by a BIONJ analysis and with cal albimana generally distributed in Europe and bootstrap resampling of 1000 pseudoreplicates for the Minor Asia) and morphotype B (the morphologi- node support assessment. BI was implemented in cal divergent populations from the Carpathians mrBayes (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck, 2003) using a Bayesian Markov-Chain and Rhodope Mountains). Monte-Carlo (B/MCMC) method. Two parallel analyses were ran for 500,000 generations using 4 chains (1 cold, 3 Geometric morphometrics heated) and trees were sampled every 100 generations. Centroid wing sizes were significantly different The first 25% of sampled trees were discarded as burn-in. (Wilcox-test, n=87, W=104, p<0.001) in the case of The B/MCMC tree was visualized with FigTree, v1.4 morphotype A (mean centroid size=7.7) and mor- (http://tree.bio.ed.ac.uk/software/figtree/). photype B (8.84). The PCA of wing shape variation

showed that the first two components explained Results 47.12% of the total shape variation (PC1: 31.18%, PC2: 15.94%) (Fig. 3A). The first ten accounted for Our morphometric data show two highly diver- 85.97% of the total variance. CVA, which maxi- gent morpho-groups within the widespread P. al- mized differentiation among groups and

Figure 3. A: Scatter plot from the Principal Component Analysis, based on position of 20 wing landmarks. Black points: morphotype A; gray points: morphotype B. B: Discriminate Analysis based on 19 measured character on male genitalia. Black points: morphotype A; gray points: morphotype B. C: Scatter plot from the Canonical Variate Analysis, based position of 20 wing landmarks. Dark gray column: morphotype A; light gray column: morphotype B. D: Column chart of size of wing length and width, black column: mor- photype A; gray column: morphotype B.

Morphologies tells more than molecules in the case of the European widespread 309

separated the two morphotypes without overlap lia, with only the size of character D3 smaller than (Mahalanobis distances= 6.3722, Procrustes dis- morphotype A. Discriminat Analysis (DA) was tances= 0.0356) (Fig. 3C). Significant shape dis- used to test the separation of the two divergent crimination was found (n=87, t2=863.4465, moprhotypes. The first two linear discriminates p<0.0001), when Discriminant Function Analysis explained 91.94% of the variance among the two (DFA) with 10000 replicates was used. The result groups; thus the two morphotypes were well showed that 100% (50/50 specimens) of morpho- separated by DA based on size of genitalia struc- type B and 97,3% (36/37) of morphotype A were tures (Fig. 3B). correctly classified. When using DFA with leave- one-out cross-validation 94% (47/50) of morpho- Molecular analysis type B and 94,6% (35/37) of morphotype A were The COI alignment was 658 base-pairs (bp) long grouped correctly. with a total number of 575 sites after excluding gaps and missing data. Of the 162 variable posi- Linear morphometrics tions, 161 were parsimony informative, resulting Specimens of morphotype B had significantly in 16 haplotypes with 0.8959 haplotype diversity. longer (Wilcox-test, n=93, df=196.5, p<0.0001) and When sequences of morphotype A were analyzed wider (Wilcox-test, n=93, df=172, p<0.0001) wings from a total of 576 sites, 106 were polymorphic than specimens of morphotype A (Fig. 3D). The and 101 parsimony informative, resulting in 9 hap- differences between the two species were based on lotypes with 0.8115 haplotype diversity. 19 measured characters. We used Wilcox-test The ML and BI analysis resulted in slightly when data did not show normal distribution and different tree topologies. In the case of the ML variances were equal; Welch’s test when the data tree, two haplotypes of morphotype A represent- showed normal distribution but variances were ing individuals from the United Kindom and Po- not equal; and Mood’s median test was used when land were linked to sequences of the outgroup data did not show normal distribution and equal species Bittacomorpha clavipes. One haplotype variance. We found significant differences in 16 shared by both morpho-groups form a clade with measured characters. Only the size of characters sequences of Ptychoptera contaminata without sig- D1, A2 and A3 did not differ significantly. The nificant bootstrap (BP) support. A third clade was used test, the test parameters, mean size and stan- formed by sequences of both morphotypes with- dard deviance of each character are shown in Ta- out any further lineage differentiation (Fig. 4). In ble 2. Morphotype B specimens had larger genita- contrast the BI tree shows a well-supported line-

Table 2. Statistical values of the measured characters on parts of male genitalia in the case of Ptychoptera albimana (morphotype A) and Ptychoptera incognita sp. n. (morphotype B).

Morphotype A Morphotype B Carac-ters Test Statistical analysis (n=101) Mean (SD) Mean (SD) D1 Wilcoxon test df = 871.5, p=0.06968 0.27 (1SD: 0.03) 0.28 (1SD: 0.03) D2 Wilcoxon test df = 417.5, p<0.001 0.93 (1SD: 0.07) 1.01 (1SD: 0.07) D3 Wilcoxon test df = 1897.5, p<0.001 0.34 (1SD: 0.03) 0.28 (1SD: 0.03) D4 Welch's t test t= -10.4241, df=56.114, p<0.001 0.24 (1SD: 0.04) 0.34 (1SD: 0.04) D5 Wilcoxon test df = 536, p<0.001 0.57 (1SD: 0.06) 0.64 (1SD: 0.06) V1 Welch's t test t= -10.7283, df=69.372, p<0.001 0.31 (1SD: 0.03) 0.40 (1SD: 0.03) V2 Mood's median test p= 0.0059 0.14 (1SD: 0.05) 0.22 (1SD: 0.05) V3 Wilcoxon test df = 90, p<0.001 1.32 (1SD: 0.08) 1.55 (1SD: 0.08) S1 Welch's t test t= -7.7386, df=67.7, p<0.001 0.27 (1SD: 0.04) 0.34 (1SD: 0.04) S2 Welch's t test t= -10.4888, df=48.842, p<0.001 0.23 (1SD: 0.02) 0.30 (1SD: 0.02) S3 Mood's median test p<0.001 0.65 (1SD: 0.06) 0.74 (1SD: 0.06) T1 Wilcoxon test df = 407, p<0.001 0.95 (1SD: 0.09) 1.06 (1SD: 0.09) T2 Welch's t test t= -14.085, df=45.564, p<0.001 0.16 (1SD: 0.01) 0.24 (1SD: 0.01) T3 Wilcoxon test df = 68, p<0.001 0.31 (1SD: 0.02) 0.39 (1SD: 0.02) T4 Wilcoxon test df = 398.5, p<0.001 0.39 (1SD: 0.06) 0.46 (1SD: 0.06) A1 Wilcoxon test df = 782.5, p =0.01396 1.06 (1SD: 0.09) 1.08 (1SD: 0.09) A2 Wilcoxon test df = 870.5, p = 0.06859 0.86 (1SD: 0.08) 0.89 (1SD: 0.08) A3 Wilcoxon test df = 1212.5, p = 0.51228, 1.12 (1SD: 0.11) 1.10 (1SD: 0.11) H1 Welch's t test t= -5.0903, df= 59.438, p<0.001, 1.17 (1SD: 0.09) 1.27 (1SD: 0.09) 310 E. Török et al.

Figure 4. Maximum Likeli- hood (ML) phylogenetic tree of Ptychoptera albimana (morphotype A) and Ptychoptera incognita sp. n. (morphotype B) with Bitta- comorpha clavipes, Ptychop- tera contaminata and Ptychoptera scutellaris as outgroups.

age of morphotype A specimens from the United ent, as well. Individuals belonging to the geo- Kingdom and Poland. Further topology of the tree graphically widespread group (morphotype A) is unresolved, showing a polytomy with three were generally present at lower altitudes, between branches representing P. contaminata, the haplo- 10-350 m and were identified in various habitats type shared by both morpho-groups and a clade from muddy lowland streams with high FPOM representing sequences of both morphotypes (Fig. content, to shrubs near marshy ponds. Specimens 5). belonging to morphotype B represent a more stenotopic group and are restricted to the Carpa- thians and Rhodope Mountains at higher eleva- Discussion tions between 450-1400 m, frequently in deep val- leys that also host a number of important glacial We used a combination of different morphometric relic species, like Tofieldia calyculata, Cladium maris- methods to test taxonomic hypotheses in the case cus, Achillea impatiens, Drosera rotundifolia, Ligularia of the widespread P. albimana. The presence of two sibirica, Cnidium dubium, Polemonium caeruleum, highly divergent morphological structures among Pedicularis sceptrum-carolinum and so on (Nyárády P. albimana were statistically well supported by 1941, Săvulescu 1976, Macalik 2011). This situation linear and also by geometric morphometry. The was identified in the case of the populations from present study is the first moprhometric analysis of Morii Valley (456 m), the Retezat Mountains (1450 a member belonging to the Ptychopteridae family. m), Giurgeu Mountains (907 m) and Rodna Moun- The presently identified morphological diversity tains (720 m) in the Eastern Carpathians. Such of P. albimana was completely overlooked in the morphologically distinct populations are likely to existing literature. However, quite recently be remnants of once formerly more widespread Ujvárosi et al. (2011) noticed the possibility of Carpathian populations that most probably sur- some unrecognized taxa among P. albimana based vived Last Glacial Maximum in one or more Car- on important morphological differences detected pathian refugia, isolated from the rest of the Euro- in populations from the Carpathian and Balkan pean morphotype A populations. Similar deep areas. morphological divergences were identified in a se- The two morpho-groups identified in the pre- ries of other widespread European aquatic insects, sent study are different along an altitudinal gradi- like Pediciidae (Diptera) (Ujvárosi et al., 2010) or

Morphologies tells more than molecules in the case of the European widespread 311

Figure 5. Bayesian inference (BI) phylogenetic tree of Ptychoptera albimana (morphotype A) and Ptychoptera incognita sp. n. (morphotype B) with Bittacomorpha clavipes, Ptychoptera contaminata, and Ptychoptera scutellaris as outgroups.

Rhyacophilidae (Trichoptera) (Bálint et al. 2008), as demonstrated in a series of recent studies (Pauls but also in Lepidoptera (Schmitt 2007), which are et al. 2009). frequently related to range contraction and isola- As previously observed in some caddis tion during the Pleistocene climate change (Bálint cases in the Carpathians (Pauls et al. 2009), “DNA et al. 2011). In the case of the stream dwelling cad- barcoding” did not support the morphological di- dis fly Drusus romanicus, morphological variability vergences in the case of P. albimana. Similar con- is not associated with corresponding molecular trasting results were frequently documented variability, because of a recent speciation event among Diptera and suggest limited utility of DNA where genetic introgression or hybridization barcoding for a generalized identification system might frequently occur (Pauls et al. 2009). in this large group of insects (Pfenninger & The presence of a single population identified Schwenk 2007, Skevington et al. 2007, Renaud et in the Rhodope Mountains belongs to morphotype al. 2012, Ashfaq et al. 2014). Multiple reasons were B suggests a more widespread range of this relic- suggested for the series of incongruence between like taxon, and reflects a close relationship be- molecular and morphological data. There is wide- tween the Carpathian and Balkan glacial refugia, spread assumption that intraspecific variation is 312 E. Török et al. usually lower than 1% and congeneric species re- m, 46.7019° N 23.5885° E, 12.04.2013, leg. Edina veal a variation generally greater than 2% (Avise Török. Paratypes 2♂♂: Poiana Mărului, Bistra Mă- 2000, Hebert et al. 2003). This rule, the so called rului Valley, 1450 m, 45.3483° N 22.6462° E, “barcoding gap”, could be the result of a poor se- 25.05.2014, leg. Levente-Péter Kolcsár. 1♂: Poiana quence quality or insufficient sampling (Wiemers Mărului, Bistra Mărului Valley, 1450 m, 45.3482° & Fiedler 2007). Additionally there are other, more N 22.6325° E, 02.06.2015, leg. Levente-Péter Kol- general concerns regarding the utility of the seem- csár. Other material: see Table. 1. ingly arbitrary 2% rule and there is now a broad opposition in the literature, which calls the so- Diagnosis called “barcoding gap” into question, since intras- Ptychoptera incognita sp. n. is similar to the sibling pecific variation may overlap interspecific diver- species P. albimana by coloration, shape of antenna gence (Shearer & Coffroth, 2008). Incomplete line- and numbers of antennal segments, but they differ age storage and historic introgression with similar in a series of details on genitalia (in Fig. 6, differ- mtCOI sequences was identified in the case of two ences are marked by arrows) and wing design closely related, partially sympatric, but morpho- (Fig. 6, differences are spotted wings). logically divergent Trichoptera in Eastern Europe (Pauls et al. 2009). DNA barcoding cannot reliably Description identify species in Calliphoridae (Diptera) because Male. Head: shining black, face yellowish brown, of the presence of the maternally transmitted Wol- basal of maxillary palpus and first segment yel- bachia associated with mitochondria and in this lowish brown, remaining four segments brown to case assignment of unknown individuals to spe- black. Scape, pedicel and antennal flagellomeres cies is not possible in 60% of the cases (Wiemers & are black, the first flagellomere twice as long as the Fiedler 2007). second flagellomere. Although there are a series of recent work Thorax: dorsally and laterally dark metallic which suggest the utility of DNA barcodes to dis- black, only pronotum and membranous parts cover and detect new species and to infer phy- around first spiracular disc are yellowish, scutel- logenetic relationships (Pauls et al. 2010, Renaud lum partly yellowish, mostly in middle part. Hind et al. 2012, Asfaq et al. 2014), our results suggest coxa is black, fore and mid coxae are yellow with an important conflict between morphological and some black spots. Trochanter and femur are molecular data. Our molecular datasets were in- brown, apical parts of femur black. Tibia and tar- valuable in assisting taxonomic decision-marking someres black, except the hind leg first tarsus in the case of the two morphologically divergent which is light brown to brown. Halteres are yel- P. albimana groups. A combination of mitochon- low on basal and brown in apical part. Length of drial and nuclear markers or phylogenetic analy- the wing 9.33 (1SD: 0.46) mm and width 3.01 (1SD: ses based on multigene analyses is highly recom- 0.16) mm. Wing with black spots in intersection of mended to resolve this conflicting situation. wing veins (Fig. 6J). Despite these incongruences between our Abdomen: shining black with pale spot on 3th morphological and molecular results, the major tergite and sternite, genitalia pale to brown color. differences between these two morpho-goups, Male genitalia (Fig. 6E, F, G, H, L): The most well supported by statistical analyses, suggest iso- distinctive characters from P. albimana (Fig. 6A, B, lation among the groups investigated, well differ- C, D, I, K), which are indicated with arrows, ap- entiated at the species level. For this reason, a new pear as follows: shape of the plate on ventral parts species will be described in the present study rep- of 9th tergite; in the case of P. incognita sp. n. it is resenting populations from the Carpathians and more developed and widened (Fig. 6F) than of P. the Rhodope Mountains (moprhotype B in the albimana; shape of lateral projections on posterior present work). parts of tegmen and the shape of anterior part of tegmen; in the case of P. incognita sp. n., it does not invaginate in middle part (Fig. 6G). Other charac- Description of Ptychoptera incognita sp. n. ters can vary greatly in both species. Török, Kolcsár et Keresztes Etymology: Name of incognita is adopted here because the presence of this new species was Type material completely overlooked in the previous literature. Holotype: 1♂ Romania, Feleacu, Morii Valley, 680 Female: We did not find clear differences be-

Morphologies tells more than molecules in the case of the European widespread 313

Figure 6. Parts of male genitalia and wing of Ptychoptera albimana (morphotype A) (A-D, K, I) and Ptychoptera incognita sp. n. (morphotype B) (E-H, L, J). A, E: dorsal view of 9th tergite and surstyle. B, F: ventral view of 9th tergite and surstyle. C, G: ventral view of style and tegmen. D, H: ventral view of hypandrium. I. J: right wing. K, L: ventral view of aedoeagus. The most distinctive characters between two species indicated with arrows.

tween P. albimana and P. incognita sp. n. females. the Romanian Carpathians and Rhodope Moun- Only small difference could be detected in the col- tains (Bulgaria). oration of hind tarsi. P. albimana has hind tarsus The two closely related species are sympatric white to white-pale in coloration, while P. incog- in Romania and Bulgaria, but there are important nita sp. n. females has brown hind tarsus. ecological differences between these two forms. P. Larvae: unknown albimana was identified at low altitude between 10- 350 m. P. incognita sp. n. were consequently col- Distribution lected at altitude between 450-1450 m (Table 2). Our data suggests that this new taxa is present in

314 E. Török et al.

Acknowledgements. We would like to acknowledge the Freeman, P. (1950): Family Ptychopteridae. In: Handbook for the work of all members of Transylvanian Dipterological Identification of British Insects IX: 2. Diptera, . Working Group; the collectors, A. Brink, Wolfram Graf London. Gouy, M., Guindon, S., Gascuel, O. (2010): SeaView version 4: a and Miklós Bálint, for sending materials; Gábor Dániel multiplatform graphical user interface for sequence alignment Lengyel for loan material from the Diptera collection of and phylogenetic tree building. Molecular Biology and the Hungarian Natural History Museum, Budapest; Evolution 27(2): 221-224. Thomas Pape, Peter Zwick, and Chen W. Young for Guindon, S., Gascuel, O. (2003): A simple, fast, and accurate identification and useful comments; and Dr. Zsolt Pénzes algorithm to estimate large phylogenies by maximum likelihood. Systematic biology 52(5): 696-704. for guidance. The work was financed partly by IDEI grant Hausdorf, B. (2011): Progress toward a general species concept. nr. PN-2-ID-PCE-2012-4-0595 of the Romanian Evolution 65: 923–931. Government and co-financed by the European Social Hebert, P.D.N., Cywinska, A., Ball, S.L., de Waard, J.R. (2003): Fund in the framework of TÁMOP-4.2.4.A/2-11/1-2012- Biological identifications through DNA barcodes. Proceedings. 0001 “National Excellence Program”. During preparation Proceedings of the Royal Society of London B: Biological of the manuscript Dénes A.L. and Kolcsár L.P. received Sciences 270(1512): 313–321. Hofmann, W. (1990): Weichselian chironomid and cladoceran financial support from the Collegium Talentum assemblages from maar lakes. Hydrobiologia 214: 207-2011. scholarships, Hungary Additional funding comes from Huelsenbeck, J.P., Ronquist, F. (2001): MRBAYES: Bayesian the research project number P 23687-B17, Pl: J. Waringer, inference of phylogenetic trees. Bioinformatics 17(8): 754-755. funded by the Austrian Science Fund (FWF). Istonia, A.G., Kiknadze, I.I., Gunderina, L.I. (2009): Chromosomal variability in natural populations of Chironomus cingulatus Meigen (Diptera, Chironomidae). Cell and Tissue Biology 3: 149- 161.

Jong de, H. (1998): In search of historical biogeography patterns in References western Mediterranean terrestrial fauna. Biological Journal of

the Linnean Society 65: 99-164. Andersson, H. (1997): Diptera Ptychopteridae, Phantom Crane Jong de, H., Oosterbroek, P., Gelhaus, J., Reusch, H., Young, C. . In: Nilsson A.N. (ed.), Aquatic Insects of North Europe. (2008): Global diversity of crane flies (Insecta, Diptera: Tipulidea Apollo Books, Stenstrup, Denmark. or Tipulidae sensu lato) in freshwater. Hydrobiologia 595: 457- Ashfaq, M., Hebert, P.D., Mirza, J.H., Khan, A.M., Zafar, Y., Mirza, 467. M.S. (2014): Analyzing mosquito (Diptera: Culicidae) diversity Kehlmaier, C., Almeida, J.M. (2014): New host records for in Pakistan by DNA barcoding. PloS one 9(5): e97268. European Acroceridae (Diptera), with discussion of species Avise, J.C. (2000): Phylogeography: the History and Formation of limits of Acrocera orbiculus (Fabricius) based on DNA-barcoding. Species. Harvard University Press, Cambridge. Zootaxa 3780(1): 135-152. Bálint, M., Barnard, P. C., Schmitt, T., Ujvárosi, L., Popescu, O. Klingenberg, C.P. (2011): MorphoJ: an integrated software package (2008): Differentiation and speciation in mountain streams: a for geometric morphometrics. Molecular Ecology Resources 11: case study in the caddisfly Rhyacophila aquitanica (Trichoptera). 353–357. Journal of Zoological Systematics and Evolutionary Research Kotlik, P., Berrebi, P. (2002): Genetic subdivision and biogeography 46(4): 340-345. of the Danubian rheophilic barb Barbus petenyi inferred from Bálint, M., Domisch, S., Engelhardt, C.H.M., Haase, P., Lehrian, S., phylogenetic analysis of mitochondrial DNA variation. Sauer, J., Nowak, C. (2011): Cryptic biodiversity loss linked to Molecular Phylogenetics and Evolution 24: 10-18. global climate change. Nature Climate Change 1(6): 313-318. Krzemiński, W. (1986): Ptychopteridae of Poland (Diptera, Beebee, T.J.C., Rowe, G. (1999): Microsatellite analysis of natterjack Nematocera). Polskie Prismo Entomologiczne 56: 105-132. toad Bufo calamita Laurenti populations: consequences of Krzemiński, W., Zwick P. (1993): New and little known dispersal from a Pleistocene refugium. Biological Journal of the Ptychopteridae (Diptera) from the Palaearctic Region. Aquatic Linnean Society 69: 367-381. Insects 15: 65-87. Bertone, M.A., Courtney, G.W., Wiegmann, B.M. (2008): Lukashevich, E.D. (2012): Phylogeny of Ptychopteroidea (Insecta: Phylogenetics and temporal diversification of the earliest true Diptera). Paleontological Journal 46: 476–484. flies (Insecta: Diptera) based on multiple nuclear genes. Macalik, K. (2011): Növényritkaságok Szenéte környékén. pp. 25 – Systematic Entomology 33: 668-687. 36. In: Markó, B., Sárkány-Kiss, A. (eds.): Gyergyói – medence: Bookstein, F.L. (1997): Morphometric Tools for Landmark Data: Egy mozaikos táj természeti értékei, Kolozsvári Egyetemi Kiadó, Geometry and Biology. Cambridge University Press, Kolozsvár. Cambridge. Malhotra, A., Thorpe, R.S. (2004): Maximizing information in Brendonk, T.U., Spitze, K. (2006): Linking lakes? The population systematic revisions: a combined molecular and morphological genetic structure of Chaoborus flavicans. Journal of Plankton analysis of a cryptic green pitviper complex (Trimeresurus Research 28: 1015-1025. stejnegeri). Biological Journal of the Linnean Society 82: 219–235. Brendonk, T.U., Spitze, K., Kerfoot, W.C. (2009): Ephemeral Michailova, P., Krastanov, B., Matena, J. (2008): Chromosomal metapopulations show high genetic diversity at regional scales. polymoprhism of Chironomus plumosus (Linnaeus, 1858) Ecology 90: 2670-2675. (Diptera, Chironomidae) from Bulgaria (Pazardjik region) and Brooks, S.J., Birks, H.J.B. (2000): Chironomid-inferred late glacial Czech Republic (Ceske Budejovice region). Comparative and early Holocene mead July air temperatures for Krakenes Cytogenetics 2: 57-65. Lake, western Norway. Journal of Paleolimnology 23: 77-89. Milankov, V.E., Francuski, L.J., Ludoški, J.A., Ståhls, G.U., Vujic, A. Darriba, D., Taboada, G.L., Doallo, R., Posada, D. (2012): (2010): Genetic structure and phenotypic diversity of two JModelTest 2: more models, new heuristics and parallel northern populations of Cheilosia aff. longula (Diptera: computing. Nature Methods 9: 772-772. Syrphidae) has implications for evolution and conservation. Dayrat, B. (2005): Towards integrative taxonomy. Biological Journal European Journal of Entomology 107: 305–315. of the Linnean Society 85: 407–415. Milankov, V., Gunilla, S., Stahls, G., Vujič, A. (2008): Genetic Deliné-Draskovits, Á. (1983): Redős szúnyogok – Ptychopteridae. characterization of the Balkan endemic species Merodon Fauna Hungariae, XIV. Diptera I. Akadémiai Kiadó, Budapest. desuturinus (Diptera, Syrphidae). European Journal of Entomology 105: 191-204. Morphologies tells more than molecules in the case of the European widespread 315

Milankov, V., Stamenkovič, J., Ludoški, J., Stahls, G., Vujič, A. Schutze, M.K., Krosch, M.N., Armstrong, K.F., Chapman, T.A., (2005): Diagnostic molecular markers and the genetic Englezou, A., Chomič, A., Cameron, S.L., Hailstones, D., Clarke, relationship among three species of the Cheilosia canicularis A.R. (2012): Population structure of Bactrocera dorsalis s.s., B. group (Diptera, Syrphidae). European Journal of Entomology papayae and B. philippinensis (Diptera: Tephritidae) in southeast 102: 125-131. Asia: evidence for a single species hypothesis using Nakamura, T. (2012): A description of a new species of Ptychoptera mitochondrial DNA and wing-shape data. BMC Evolutionary Meigen, 1803 from the Shirakami Mountains, Honshu, Japan Biology 12: 130-145. and some notes on distributions of Japanese species (Diptera, Shearer, T.L., Coffroth, M.A. (2008): DNA BARCODING: barcoding Ptychopteridae). Shirakami-Sanchi: Bulletin of the Shirakami corals: limited by interspecific divergence, not intraspecific Institute for Environmental Sciences. Hirosaki University 1: 15- variation. Molecular Ecology Resources 8(2): 247-255. 18. Skevington, J.H., Kehlmaier, C., Stahls, G. (2007): DNA Barcoding: Nakamura, T., Saigusa, T. (2009): Taxonomic study of the family Mixed results for big-headed flies (Diptera: Pipunculidae). Ptychopteridae of Japan (Diptera). Zoosymposia 3: 273-303. Zootaxa 1423: 1-26. Nyárády, E.G. (1941): Kolozsvár és környékének flórája. Erdélyi Stubbs, A. (1993): Provisional atlas of the ptychopterid craneflies Nemzeti Múzeum Növénytára, Kolozsvár. (Diptera: Ptychopteridae) of Britain and Ireland. Biological Orendt, C., Mischke, U., Nixdorf, B., Brooks, S. (2009): Subfossil Records Centre Institute of Terrestrial Ecology, Huntingdon. chironomids in shallow lakes of northern Germany. Tjeder, B. (1968): Notes on the Scandinavian Ptychopteridae, with Lauterbroenia 68: 59-70. description of a new species (Diptera). Opuscula Entomologica Padial, J.M., Miralles, A., De la Riva, I., Vences, M. (2010): Review: 33: 73-79. The integrative future of taxonomy. Frontiers in Zoology 7: 1-14. Ujvárosi, L., Bálint, M., Mészáros N., Popescu O. (2009): Genetic Paramonov, N.M. (2012): Ptychopteridae, a family of flies (Diptera) diversity with morphological imprints among Pedicia (Amalopis) new to the Philippine Islands with the description of a new occulta (Meigen, 1830) (Diptera, Pediciidae) populations in the species. Zootaxa 3682: 584-588. Carpathian area: Preliminary results. Lauterbornia 68: 47–58. Paramonov, N.M., (2013): New records of Ptychopteridae (Diptera) Ujvárosi, L., Bálint, M., Schmitt, T., Popescu, O., Ujvárosi T. (2010): from Europe, North Africa and Asia Minor. Studia Divergence and speciation in the Carpathians area: patterns of Dipterologica 20: 279-283. morphological and genetic diversity of the crane fly Pedicia Papp, L., Földváry, M. (2007): Középhegységeink patakjainak occulta (Diptera: Pediciidae). Journal of the North American egyedülálló légyfaunája. pp. 369-374 In: Forró, L. (ed.), A Benthological Society 29: 1075–1088. Kárpát-medence állatvilágának kialakulása. Magyar Ujvárosi, L., Kolcsár,P., Török, E. (2011): An annotated list of Természettudományi Múzeum, Budapest. Ptychopteridae (Insecta, Diptera) from Romania, with notes on Pauls, S.U., Blahnik, R.J., Zhou, X., Wardwell, C.T., Holzenthal, the individual variability of Ptychoptera albimana (Fabricius, R.W. (2010): DNA barcode data confirm new species and reveal 1787). Entomologica Romanica 16: 39-45. cryptic diversity in Chilean Smicridea (Smicridea) (Trichoptera: Ujvárosi, L., Bálint, M. (2012): Discovery of the second European Hydropsychidae). Journal of the North American Benthological Amalopis species: an integrative survey of the widespread Pedicia Society 29: 1058–1074. (Amalopis) occulta (Meigen, 1830) (Insecta, Diptera, Pediciidae). Pauls, S.U., Theissinger, K., Ujvárosi, L., Bálint, M., Haase, P. (2009): Zootaxa 3189: 1-28. Patterns of population structure in two closely related, partially Vinogradova, E.B., Shaikevich, E.V., Ivanisky, A.V. (2007): A study sympatric caddisflies in Eastern Europe: historic introgression, on the distribution of the Culex pipiens complex (Insecta, limited dispersal, and cryptic diversity. Journal of the North Diptera, Culicidae) mosquitoes in the European part of Russia American Benthological Society 28: 517–536. by molecular methods of identification. Comparative Pauls, S.U., Lumbsch, H.T., Hasse, P. (2006): Phylogeography of the Cytogenetics 1: 129-138. montane caddisfly Drusus discolor: evidence for multiple refugia Wagner, R., Barták, M., Borkent, A., Courtney, G., Goddeeris, B., and periglacial survival. Molecular Ecology 15: 2153–2169. Haenni, J.P., Knutson, L., Pont, A., Rotheray, G.E., Rozkošný, R., Peus, F. (1958): Liriopeidae. pp: 10-44. In: Lindner, E. (ed), Die Sinclair, B., Woodley, N., Zatwarnicki, T., Zwick, P. (2007): Fliegen der palaearktischen Region. III, Schweizerbart, Global diversity of dipteran families (Insecta Diptera) in Stuttgard. freshwater (excluding Simulidae, Culicidae, Chironomidae, Pfenninger, M., Schwenk, K. (2007): Cryptic species are Tipulidae and Tabanidae). Hydrobiologia 595: 489–519. homogeneously distributed among taxa and biogeographical Wiemers, M., Fiedler, K. (2007): Does the DNA barcoding gap regions. BMC Evolutionary Biology 7(1): 121. exist?–a case study in blue butterflies (Lepidoptera: Lycaenidae). Renaud, A.K., Savage, J., Adamowicz, S.J. (2012): DNA barcoding Frontiers in Zoology 4(8): 1-16. of Northern Nearctic Muscidae (Diptera) reveals high Yeates, D. K., Wiegmann, B.M. (2005): Phylogeny and evolution of correspondence between morphological and molecular species Diptera: recent insights and new perspectives. pp. 14-44. In: limits. BMC Ecology 12(1): 24-39. Yeates, D. K., Wiegmann, B. M. (eds.), The evolutionary biology Ronquist, F., Huelsenbeck, J.P. (2003): MrBayes 3: Bayesian of flies. Columbia University Press, New York. phylogenetic inference under mixed models. Bioinformatics Zelditch, M.L., Swiderski, D.L., Sheets H.D., Fink, W.L. (2004): 19(12): 1572-1574. Geometric morphometrics for biologists: a primer. Elsevier Rozkošný, R. (1997): Family Prychopteridae. pp: 291-296. In: Papp. Academic Press, San Diego. L. Darvas, B. (eds.): Contributions to a Manual of Palaearctic Zitek-Zwyrtek, K. (1971): Czechoslovak species of the family Diptera, Volume 2. Nematocera and Lower Brachicera. Science Ptychopteridae (Diptera). Acta Entomologica Bohemoslovaka Herald, Budapest. 68: 416-426. Săvulescu, T. (1976): Flora Republicii Populare Romîne VIII. Vol. Zwick, P., Starý, J. (2003): Ptychoptera delmastroi sp. n. (Diptera, Editura Academiei Republicii Populare Române, Bucureşti. Ptychopteridae) from Italy. Aquatic Insects 25: 241-246. Schmitt, T. (2007): Molecular biogeography of Europe: Pleistocene cycles and postglacial trends. Frontiers in Zoology 4(11): 1-13.