Genetic variability of two ecomorphological forms of Stenus Latreille, 1797 in Iran, with notes on the infrageneric classification of the genus (Coleoptera, Staphylinidae, Steninae).

In this study, the genetic diversity of Iranian populations of two widespread Stenus species representing two ecomorphological forms, the "open living species" Stenus erythrocnemus Eppelsheim, 1884 and the "stratobiont" Stenus callidus Baudi di Selve, 1848, is presented using data from a fragment of the mitochondrial COI gene. We evaluate the mitochondrial cytochrome oxidase I haplotypes and the intraspecific genetic distance of these two species. Our results reveal a very low diversity of COI sequences in Stenus erythrocnemus in contrast to Stenus callidus. Moreover, the COI based phylogeny of a selection of Iranian Stenus support the monophyly of some species groups of Stenus proposed by Puthz (2008) and contradicts the traditional infrageneric classification.

Introduction

Fast mutation rate and lack of recombination as well as its easy amplification and sequencing make COI a useful marker for the study of phylogeny, geographic variation and population genetics as well as species identification (Hebert et al. 2003a, b; Qian et al. 2014: 11). Many studies have demonstrated that mtDNA-COI can be used for population genetics (e.g. Szalanski et al. 2010: 8). Hajibabaei et al. (2007: 171) point out that DNA barcoding offers significant implications for the understanding of the genetic diversity of species. Here, we apply this method in the rove beetle genus Latreille, 1797 to test the infraspecific genetic variation of representatives of two distinct ecomorphological forms and the validity of the traditional subgeneric concept of the genus.

is well-known for its unique prey-capture behavior (e.g. Betz 1996: 15–34). The eversible labium, an apomorphy, and the variability of the tarsal structures seem to be responsible for the enormous radiation in this genus (Betz 2002: 1097). The labial features are involved in catching prey in a sudden manner despite the limited reaction ability of the beetle (Betz 1999: 1708). The variable tarsal morphology among the members of this genus also has adaptive values which are in accord with their habitat preferences (Betz 2006: 413–414). With about 2674 species (Puthz, unpublished), is one of the species-richest genera of animals in the world (Puthz 2012: 286). The members of this rove beetle clade mostly dwell in humid places such as river banks, swamps, bogs and wet grasslands. The multifunctional secretion of the pygidial glands is species-specific and acts as a survival factor against predators. This character has been used in illuminating several evolutionary trends (Schierling et al. 2013: 48, 51) and presumably is a character adaptive to the habitat where the species live (Lang et al. 2015: 22).

In , two major ecomorphological forms can be distinguished, which Kastcheev and Puthz (2011: 454) termed “open-living species” with longer legs and on average bigger bodies (Figure 1), that live in habitats with less dense, often sparse vegetation such as sandy or clayey banks, and “stratobionts” with shorter legs and compact body (Figure 2), which inhabit dense vegetation structures and organic litter. Both forms are moreover distinguished by their dispersal ability, because - unlike the open-living species - there is the evolutionary tendency in stratobionts towards flightlessness. Similar morphological adaptations were already described for many rove-beetle clades such as the paederine subtribe Mulsant & Rey, 1878 (Frisch et al. 2002: 30). The addressed morphological characters determine the ability of the organism to colonize particular habitats and to use their resources (Betz 2006: 413). This relation between morphological features of species and ecological characteristics of habitats seems to be descriptive for niche selection. In Iran, 68 species were recorded (Serri and Frisch 2016: 18), among which Eppelsheim, 1884 and Baudi di Selve, 1848 are the most widespread across the country and were found in most provinces of Iran. According to Kastcheev and Puthz (2011: 454), is an open-living species and a stratobiont. Based on Iranian populations of these species, we tested the hypothesis that open-living species show a lower infraspecific genetic diversity than stratobionts owing to their higher dispersal ability.

Figures 1–4.

1 Eppelsheim, 1884. 2 Baudi di Selve, 1848. 3 Haplotype network for DNA sequences of . The circle size shows the frequency of the haplotypes. Each dashed line represents a single mutation. 4 Haplotype network for DNA sequences of . The circle size shows the frequency of the haplotypes. Each dashed line represents a single mutation. Scale bars: 1mm.

cytochrome c oxidase subunit I

cytochrome c oxidase subunit I

was traditionally divided into subgenera according to morphological characters. Based on European species only, Rey (1884: 31) introduced the six subgenera , , , , , and . Later, Heyden (1905: 262) replaced with because of a homonymy with a genus in the . Ádám (1987: 135), however, synonymized with the older name Motschulsky, 1860, because the type species of both subgenera are considered as subjective synonyms. Therefore he introduced the new subgenus for a distinct species group of (Herman 2001: 2041), but later he (Ádám 2001: 126) replaced this name with because of primary homonymy with Walker, 1834 in the . According to Puthz (2009: 47), the genus group name Özdikmen & Darılmaz, 2008, an unnecessary replacement name for , is a junior synonym of . Puthz (2001: 35) also synonymized with s. str. based on the assumption that the tarsal characters traditionally employed for these subgenera do not define distinct monophyletic groups. In the current edition of the Catalogue of Palaearctic , Schülke and Smetana (2015: 802–847) still divided this genus into five subgenera, which are Motschulsky, 1860, Rey, 1884, Latreille, 1797, PageBreak Ádám, 2001 and Rey, 1884. Puthz (2008: 139–148) conceived that the traditional subgeneric classification does not reflect the phylogenetic affinities within this genus and thus established 157 monophyletic species groups based on a wide range of presumed apomorphic morphological features of the species included. Ryvkin (2011: 59) argued, however, that it is better not to reject the traditional subgeneric concept prior to a comprehensive phylogenetic analysis of the subfamily. To date, there are only a few molecular studies that have investigated the phylogenetic relationships among species. The first was done recently by Koerner et al. (2013). Their results supported the monophyletic groups proposed by Puthz (2008: 139–148) and moreover revealed that some species groups of Leach, 1819, the second genus of the , actually constitute a monophyletic group within . The monophyly of some species groups proposed by Puthz (2008: 141–147) was also supported by Lang et al. (2015: 21). We performed a preliminary investigation on the intra- and interspecific genetic diversity of some Iranian to test the validity of the traditional classification of this genus by sequencing the “DNA Barcode” region of the mitochondrial COI gene of these species.

Material and methods

The specimens this study is based on were collected in the framework of the first author’s research project on the diversity and biogeography of this genus in Iran (Serri and Frisch 2016), which was a part of a joint project between the Museum für Naturkunde Berlin and the Iranian Research Institute of Plant Protection on biodiversity and biogeography of selected insect taxa in Iran.

The specimens were collected in humid habitats such as river banks or grassland by hand collecting or sifting of gravelly soil, leaf litter and other phytodebris. Most specimens were killed with ethyl acetate, but some were directly fixated in 96% ethanol.

For DNA extraction, the abdomen of the larger species and the whole body of the smaller species were used and the DNA was purified by the CTAB method (Winnepenninckx et al. 1993). The was used to amplify a 5’ end fragment of the mitochondrial gene using the primer pair LCO1490 5’-GGTCAACAAATCATAAAGATATTGG -3’ and HCO2198 5’-TAAACTTCAGGGTGACCAAAAAATCA -3’ (Folmer et al. 1994). PCR was performed in 25µl volumes including 2.5 µM PCR buffer, 1µM MgCl2, 0.5 µM dNTP, 1 µM of each forward and reverse primers, 1µM of Taq polymerase and ddH2O up to 25 µl total volumes. In the PCR thermocycles, there was an initial denaturation step at 94° for 1.5 min, followed by 6 cycles of 94° (for 30 s) denaturation, 45° (1.5 min) annealing and 72° (for 1 min) extension and subsequently 35 cycles of 94° (for 30 s) denaturation, 51° (1.5 min) annealing and 72° (for 1 min) extension. The PCR terminated at 72° (for 5 min) for final extension. The PCR products were purified on a silica membrane with Macherey and Nagel Nucleospin kits following the manufacturer’s protocol. The purified PC products were sequenced using an ABI 3130 DNA PageBreakPageBreakPageBreaksequencer. All sequences were aligned manually and corrected for misreads using Bioedit version 7.0.5.3 (Hall 1999). Additional mitochondrial COI GenBank sequences of (Lacordaire, 1835) and Peyerimhoff, 1937 were included in the dataset (GenBank accession numbers KM447120 and KM451370) as PageBreakoutgroup taxa. A Maximum Parsimony Analysis was conducted with PAUP*4.0 b10 (Swofford 2002). The dataset was also analyzed in MEGA 6 (Tamura et al. 2013) with maximum likelihood using the Tamura-Nei model with uniform rates among sites. The mean p-distance within each species of and were calculated separately using the Kimura2-parameter model (Kimura 1980) in MEGA 6. The haplotype data files of the populations of each species and the polymorphisms indices were obtained in DnaSP 5.10 (Librado and Rozas 2009) and the nexus files were transferred to PopART version 1.7 (Leigh and Bryant 2015) in order to construct a haplotype network based on the TCS algorithm (Clement et al. 2002).

polymerase chain reaction

Results

The PCR amplification using LCO1490/HCO2198 primers yielded a product with a maximum length of 658 bp (excluding primers) from 91 individuals of 23 species out of a total of 157 specimens of 37 species of Iranian . The alignment was blasted against GenBank sequences and found to match with existing records of . The base composition of about 29% A, 39% T, 16% C and 16% G exhibits the common AT bias of COI.

The alignment (total of 658 bp) contained 294 variable characters, of which 246 were parsimony informative and contributed to the Analysis. The MP Analyses produced two equally parsimonious trees with a tree length of 1197 steps, CI of 0.3768, RI of 0.8564 and RC of 0.3227 (Figure 7). Node support was estimated by bootstrap using 1000 pseudoreplicates and 100 replicates. The major clades are generally well supported (see below). All obtained sequences were submitted to GenBank (accession numbers in Table 1). The maximum likelihood tree was constructed by the heuristic search with the method, gaps treatment using all sites, the tree as the initial tree and bootstrapped with 1000 replications (Figure 8). The topology obtained from ML analyses does not deviate significantly from the MP tree. Both methods reveal a high degree of genetic homogeny among different populations of and more pronounced heterogeny in . The selected populations of cluster in seven groups, but these groupings do not correspond well to the geographic distribution of the examined populations and some are not well supported in the bootstrap analysis. There are, however, some populations that form separate geographical clusters such as the populations from Kerman (specimen no. 034) with those of the Ghohrud Mountains (specimen no. 094) and the populations from Tehran Province (specimens no. 105, 107). These apparent geographical clusters are, however, not significant, because they are made up of only two populations from the same region (Figure 9). Surprisingly, the populations of from Kordestan Province show a low similarity of the COI gene and appear in different clades of the cladogram (Figures 7, 8).

Figure 7.

Strict consensus of most-parsimonious trees. Values above the branches indicate clade bootstrap support (>50) using 1000 replicates. The geographical origin of the specimens is coded by numbers behind the species name which correspond to the geographical information in Table 1.

Table 1.

The specimens used in this study with their location data and the GenBank association number of submitted sequences of COI. The specimen number codes the geographical origin of the specimens in the phylogenetic tree (Figures 7, 8).

Species	Specimen number	Collection site	GeneBank association number
Stenus alienigenus	147	Kordestan: 11 km E Sanandaj (35°20'11"N 47°09'07"E), 2100 m, 5.9.2008, leg. Serri and Frisch	KU754268
Stenus araxis	118	Ardabil: N Mt Sabalan, Gheynarjeh (38°17'18"N 47°41'22"E), 2100 m, 24.6.2008, leg. Serri	KU754251
Stenus araxis	121, 122	Esfahan: Kashan, NW Niasar, after Aznaveh (34°06'28.8"N 50°59'45.9"E), 2195 m, 19.5.2009, leg. Serri and Nasserzadeh	KU754253 KU754254
Stenus araxis	117	Hamedan: W Kabudarahang, 5 km E Goltappeh (35°12'06"N 48°14'04"E), 2210 m, 21.7.2008, leg. Serri and Nasserzadeh	KU754250
Stenus araxis	114	Kordestan: Saghez - Baneh, 27 km SW Saghez (36°08'12"N 46°02'42"E), 1600 m, 3.9.2008, leg. Serri and Frisch	KU754247
Stenus araxis	111	West Azarbaijan: W Salmas, 19 km W Kuzerash (38°11'40"N 44°33'04"E), 1960 m, 31.8.2008, leg. Serri and Frisch	KU754246
Stenus araxis	110	West Azarbaijan: Orumieh, S Silvaneh, 14 km S Ziveh (37°09'06"N 44°52'55"E), 2320 m, 1.9.2008, leg. Serri and Frisch	KU754245
Stenus cf. araxis	120	Esfahan: Natanz, S Karkas Mts, Taragh (33°24'39"N 51°46'14"E), 2580 m, 20.5.2009, leg. Serri	KU754252
Stenus cf. araxis	125	Esfahan: S Abyaneh, Bidhand (33°29'44"N 51°45'39"E), 2350 m, 18.5.2009, leg. Serri	KU754256
Stenus cf. araxis, Stenus araxis	115, 116	Tehran: Firouzkuh, Badroud (35°48'15"N 52°39'21"E), 2060 m, 5.8.2009, leg. Serri and Nasserzadeh	KU754248 KU754249
Stenus ater	136	Semnan: NE Chashm, Hikuh, Sheil, Parvar Protected Region (36°0'54"N 53°23'07"E), 1900 m, 7.8.2009, leg. Serri and Nasserzadeh	KU754264
Stenus brunnipes	151	Mazandaran: Sari, N Mohammadabad (36°10'09"N 53°14'08"E), 820 m, 30.5.2008, leg. Serri, Nasserzadeh and Pütz	KU754270
Stenus callidus	089	Chaharmahal & Bakhtiari: Ardel, Ghahrou, Tang-e Zeverdegan (31°59'10"N 50°51'23"E), 2350 m, 23.6.2009, leg. Serri	KU754233
Stenus callidus	090	Esfahan: Chadegan, W Zayandehrud Dam (32°43'08"N 50°44'20"E), 2070 m, 20.6.2009, leg. Serri	KU754234
Stenus callidus	094	Esfahan: Kashan, S Ghamsar, Ghazaan (33°42'20"N 51°23'48"E), 2220 m, 17.5.2009, leg. Serri	KU754236
Stenus callidus	045, 046	Ghazvin: 5 km E Abgarm (35°47'53"N 49°22'43"E), 1510 m, 21.6.2004, leg. Serri and Frisch	KU754199 KU754200
Stenus callidus	092	Hamedan: Eberou road, S Emamzadeh Abdollah (34°39'20"N 48°32'19"E), 2510 m, 22.7.2008, leg. Serri and Nasserzadeh	KU754235
Stenus callidus	103	Hamedan: Shahrestaneh (34°42'56"N 48°22'21"E), 2220 m, 23.7.2008, leg. Serri and Nasserzadeh	KU754240
Stenus callidus	031, 033	Hormozgan: Siahu, Talgerdo road, Bangolan (27°50'03"N 56°28'27"E), 890 m, 19.4.2006, leg. Serri and Frisch	KU754193 KU754194
Stenus callidus	034	Kerman: Baft, 6 km N Rabor (29°20'28"N 56°50'47"E), 2640 m, 4.5.2007, leg. Serri and Frisch	KU754195
Stenus callidus	084	Khuzestan: Baghmalek, Chamkureh (31°31'42"N 49°51'55"E), 670 m, 27-28.4.2009, leg Serri	KU754231
Stenus callidus	079–082, 085, 086	Kordestan:11 km E Sanandaj (35°20'11"N 47°09'07"E), 2100 m, 5.9.2008, leg. Serri and Frisch	KU754224 KU754225 KU754226 KU754227 KU754230 KU754231
Stenus callidus	087	Kordestan: 7 km S Ghorveh, Veihaj (35°06'34"N 47°45'54"E), 2060 m, 5.9.2008, leg. Serri and Frisch	KU754232
Stenus callidus	098, 099	Kordestan: Saghez - Baneh, 27 km SW Saghez (36°08'12"N 46°02'42"E), 1600 m, 3.9.2008, leg. Serri and Frisch	KU754237 KU754238
Stenus callidus	035, 036	Tehran: Firouzkuh road, Delichai (35°40'58"N 52°28'26"E), 2000 m, 21.5.2006, leg. Serri and Frisch	KU754196 KU754197
Stenus callidus	105–108	Tehran: Firouzkuh, Badroud (35°48'15"N 52°39'21"E), 2060 m, 5.8.2009, leg. Serri and Nasserzadeh	KU754241 KU754242 KU754243 KU754244
Stenus callidus	100	West Azarbaijan: 11 km E Takht-e Soleiman (36°36'43"N 47°18'48"E), 2280 m, 7.-8.9.2008, leg. Serri and Frisch	KU754239
Stenus callidus	083	West Azarbaijan: 2 km E Takht-e Soleiman N (36°38'05"N 47°14'07"E), 2270 m, 7.-8.9.2008, leg. Serri and Frisch	KU754228
Stenus callidus	037	Zanjan: Abbar - Gilvan (36°52'50"N 48°58'32"E), 430 m, 12.7.2006, leg. Serri	KU754198
Stenus cautus	146	Esfahan: S Abyaneh, Bidhand (33°29'44"N 51°45'39"E), 2350 m, 18.5.2009, leg. Serri	KU754267
Stenus erythrocnemus	059, 060, 062	Ardabil: N Mt Sabalan, Gheynarjeh (38°17'18"N 47°41'22"E), 2100 m, 24.6.2008, leg. Serri	KU754213 KU754214 KU754215
Stenus erythrocnemus	024	East Azarbaijan: Zijenab (Mt Sahand) (37°52'08"N 46°18'46"E), 2150 m, 8.8.2005, leg. Serri and Frisch	KU754192
Stenus erythrocnemus	134	Esfahan: Natanz, Taragh, Keshe, S Mt. Karkas (33°24'39.3"N 51°46'13.9"E), 2580 m, 17.5.2009, leg. Serri	KU754262
Stenus erythrocnemus	070	Gilan: E Masuleh (37°09'48"N 49°00'19"E), 820 m, 8.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754219
Stenus erythrocnemus	009	Kerman: Mahan road, 3 km S pass (30°11'29"N 57°25'42"E), 2430 m, 30.4.2007, leg. Serri and Frisch	KU754189
Stenus erythrocnemus	051–054	Tehran: Dizin (36°01'53"N 51°28'52"E), 2810 m, 10.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754205 KU754206 KU754207 KU754208
Stenus erythrocnemus	047–050	West Azarbaijan: SE Makou, Gharakelisa (39°05'32"N 44°32'40"E), 1860 m, 28.8.2008, leg. Serri and Frisch	KU754201 KU754202 KU754203 KU754204
Stenus erythrocnemus	055–058	West Azarbaijan: Orumieh, S Silvaneh, 14 km S Ziveh (37°09'06"N 44°52'55"E), 2320 m, 1.9.2008, leg. Serri and Frisch	KU754209 KU754210 KU754211 KU754212
Stenus erythrocnemus	064	West Azarbaijan: 18 km W Khoy, Ghotour road (38°28'45"N 44°47'08"E), 1320 m, 29.8.2008, leg. Serri and Frisch	KU754216
Stenus erythrocnemus	068, 069	West Azarbaijan: Siahcheshmeh - Khoy, Kordkandy (N 38°55'02" E44°27'40’’), 1870 m, 28.8.2008, leg. Serri and Frisch	KU754217 KU754218
Stenus erythrocnemus	071–074	West Azarbaijan: Siahcheshmeh - Khoy, W Zarabad (N 38°44'16" E44°28'10’’), 2400 m, 30.8.2008, leg. Serri and Frisch	KU754220 KU754221 KU754222 KU754223
Stenus erythrocnemus	011, 012	Yazd: Taft, Dehbala (31°35'37"N 54°07'20"E), 2550 m, 15.5.2007, leg. Serri and Frisch	KU754190 KU754191
Stenus fuscicornis	156	Mazandaran: Ramsar, Javaherdeh road, Eshkatechal (36°50'32"N 50°34'39"E), 1450 m, 6.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754272
Stenus ganglbaueri	153	Mazandaran: Baladeh, Nesen, E pass (36°14'37"N 51°27'17"E), 2960 m, 1.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754271
Stenus hypoproditor	137	Kordestan: N Divandarreh, SW Zarrineh, 5 km NW Ebrahimabad (35°59'10"N 46°52'11"E), 1960 m, 4.9.2008, leg. Serri and Frisch	KU754265
Stenus intricatus zoufali	135	East Azarbaijan: Tabriz - Marand, 9 km N Amand (38°17'18"N 46°08'46"E), 1520 m, 26.8.2008, leg. Serri and Frisch	KU754263
Stenus maculiger	133	West Azarbaijan: W Salmas, 10 km W Kuzerash (38°11'40"N 44°33'04"E), 1960 m, 31.8.2008, leg. Serri and Frisch	KU754261
Stenus martensi	166	Mazandaran: Kelardasht- Marzanabad road, (36°35'39"N 51°08'37"E), 1000 m, 3.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754279
Stenus medus	161	Mazandaran: Rineh, S Mt Damavand (35°53'56"N 52°06'29"E), 2960 m, 3.8.2009, leg Serri and Nasserzadeh	KU754276
Stenus mongolicus	138	Semnan: Shahroud, NE Mojem, Tash (36°31’N 54°42’E), 10.8.2009, leg. Serri and Nasserzadeh	KU754266
Stenus ochropus	159	Fars: SE Sepidan, Dalkhon (30°14'40"N 52°06'09"E), 2090 m, 9.5.2007, leg. Serri and Frisch	KU754275
Stenus persicus	163	Kordestan: Saghez - Baneh, 27 km SW Saghez (36°08'12"N 46°02'42"E), 1600 m, 3.9.2008, leg. Serri and Frisch	KU754277
Stenus pieperi	157	Mazandaran: S Salmanshahr (36°38'49"N 51°10'27"E), 280 m, 4.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754273
Stenus ressli	158	Mazandaran: Tonekabon, Sehezar Forest (36°32'36"N 50°49'53"E), 1090 m, 5.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754274
Stenus schah	164	Kohgiluye & Boyerahmad: N Yasuj, Sepidar, Dilgan River (30°45'03"N 51°08'07"E), 2270 m, 18.6.2009, leg. Serri	KU754278
Stenus turk	124	Esfahan: S Abyaneh, Bidhand (33°29'44"N 51°45'39"E), 2350 m, 18.5.2009, leg. Serri	KU754255
Stenus turk	126–129	Golestan: NE Kalaleh, Zav, Totlitamak village (37°29'36"N 55°46'25"E), 1240 m, , 16.10.2009, leg. Serri	KU754257 KU754258 KU754259 KU754260
Stenus viti	148	Mazandaran: Kelardasht - Marzanabad (36°35'40"N 51°08'37"E), 1000 m, 3.6.2008, leg. Serri, Nasserzadeh and Pütz	KU754269

Figure 8.

Maximum likelihood phylogram. Numbers on branches are bootstrap values (>50). The specimen codes correspond to the geographical information in Table 1. Scale shows number of substitutions per site.

Figure 9.

Distribution map of sequenced specimens of (●) and (○). Numbers are haplotype numbers (see Table 4). Sites with more than one haplotype number indicate several geographically close localities.

Maximum Parsimony

Nearest-Neighbor-Interchange

neighbor-joining

The haplotype networks for COI of and (Figures 3, 4) comprise fourteen and four haplotypes, respectively. Haplotype diversity (h) was estimated at 0.911±0.034 for and 0.267±0.107 for . The nucleotide diversity (π i) of each species was calculated as 0.01348±0.00074 for and 0.00045±0.00019 PageBreakPageBreakPageBreakfor (Table 4). In , no haplotype has an outstandingly high frequency, while has a dominant haplotype (Hap_1) found in populations of the Elburz and Zagros Mountains and the central mountain ranges.

Table 4.

Summary of genetic diversity indices in the mitochondrial COI gene segment of and .

Species	N	L	k	H	h (±standard deviation)	π (±standard deviation)	Haplotype no.: sequence(s) no.
Stenus callidus	29	658	30	14	0.911±0.034	0.01348±0.00074	Hap_1: 031, 033, 037, 085, 086 Hap_2: 034, 081 Hap_3: 035, 036 Hap_4: 045, 046 Hap_5: 079, 082 Hap_6: 080, 083, 084, 089, 090,100, 103 Hap_7: 087 Hap_8: 092 Hap_9: 094 Hap_10: 098,106 Hap_11: 099 Hap_12: 105 Hap_13: 107 Hap_14: 108
Stenus erythrocnemus	28	658	3	4	0.267±0.107	0.00045±0.00019	Hap_1: 009, 011, 012, 047, 048, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 064, 068, 069, 070, 071, 072, 073, 074, 134 Hap_2: 024 Hap_3: 049, 062 Hap_4: 060

Abbreviations: N; L; k; H; h; π.

, number of sequences

, sequence length (number of bases)

, number of variable sites

, number of haplotypes

, haplotype diversity

, nucleotide diversity

The maximum genetic distance among populations does not exceed 0.003% in and is much higher in with 0.028% (Tables 2, 3). The highest genetic distance as well as the highest haplotype diversity in the populations of was observed in the central zone of the Zagros Mountains. In , the highest genetic distance is among the populations of northwestern Iran.

Table 2.

Kimura two-parameter pairwise genetic distances between populations of .

	031	033	034	035	036	037	045	046	079	080	081	082	083	084	085	086	087	089	090	092	094	098	099	100	103	105	106	107	108
031
033	0.000
034	0.006	0.006
035	0.022	0.022	0.022
036	0.022	0.022	0.022	0.000
037	0.000	0.000	0.006	0.022	0.022
045	0.002	0.002	0.008	0.023	0.023	0.002
046	0.002	0.002	0.008	0.023	0.023	0.002	0.000
079	0.012	0.012	0.011	0.028	0.028	0.012	0.014	0.014
080	0.020	0.020	0.020	0.002	0.002	0.020	0.022	0.022	0.026
081	0.006	0.006	0.000	0.022	0.022	0.006	0.008	0.008	0.011	0.020
082	0.012	0.012	0.011	0.028	0.028	0.012	0.014	0.014	0.000	0.026	0.011
083	0.020	0.020	0.020	0.002	0.002	0.020	0.022	0.022	0.026	0.000	0.020	0.026
084	0.020	0.020	0.020	0.002	0.002	0.020	0.022	0.022	0.026	0.000	0.020	0.026	0.000
085	0.000	0.000	0.006	0.022	0.022	0.000	0.002	0.002	0.012	0.020	0.006	0.012	0.020	0.020
086	0.000	0.000	0.006	0.022	0.022	0.000	0.002	0.002	0.012	0.020	0.006	0.012	0.020	0.020	0.000
087	0.003	0.003	0.009	0.022	0.022	0.003	0.005	0.005	0.015	0.020	0.009	0.015	0.020	0.020	0.003	0.003
089	0.020	0.020	0.020	0.002	0.002	0.020	0.022	0.022	0.026	0.000	0.020	0.026	0.000	0.000	0.020	0.020	0.020
090	0.020	0.020	0.020	0.002	0.002	0.020	0.022	0.022	0.026	0.000	0.020	0.026	0.000	0.000	0.020	0.020	0.020	0.000
092	0.023	0.023	0.023	0.005	0.005	0.023	0.025	0.025	0.030	0.003	0.023	0.030	0.003	0.003	0.023	0.023	0.023	0.003	0.003
094	0.008	0.008	0.002	0.023	0.023	0.008	0.009	0.009	0.012	0.022	0.002	0.012	0.022	0.022	0.008	0.008	0.011	0.022	0.022	0.022
098	0.011	0.011	0.008	0.020	0.020	0.011	0.012	0.012	0.014	0.019	0.008	0.014	0.019	0.019	0.011	0.011	0.014	0.019	0.019	0.022	0.009
099	0.003	0.003	0.009	0.022	0.022	0.003	0.002	0.002	0.015	0.020	0.009	0.015	0.020	0.020	0.003	0.003	0.003	0.020	0.020	0.023	0.011	0.014
100	0.020	0.020	0.020	0.002	0.002	0.020	0.022	0.022	0.026	0.000	0.020	0.026	0.000	0.000	0.020	0.020	0.020	0.000	0.000	0.003	0.022	0.019	0.020
103	0.020	0.020	0.020	0.002	0.002	0.020	0.022	0.022	0.026	0.000	0.020	0.026	0.000	0.000	0.020	0.020	0.020	0.000	0.000	0.003	0.022	0.019	0.020	0.000
105	0.014	0.014	0.011	0.020	0.020	0.014	0.015	0.015	0.017	0.019	0.011	0.017	0.019	0.019	0.014	0.014	0.014	0.019	0.019	0.022	0.012	0.012	0.014	0.019	0.019
106	0.011	0.011	0.008	0.020	0.020	0.011	0.012	0.012	0.014	0.019	0.008	0.014	0.019	0.019	0.011	0.011	0.014	0.019	0.019	0.022	0.009	0.000	0.014	0.019	0.019	0.012
107	0.011	0.011	0.008	0.017	0.017	0.011	0.012	0.012	0.014	0.015	0.008	0.014	0.015	0.015	0.011	0.011	0.011	0.015	0.015	0.019	0.009	0.009	0.011	0.015	0.015	0.003	0.009
108	0.012	0.012	0.009	0.022	0.022	0.012	0.014	0.014	0.015	0.020	0.009	0.015	0.020	0.020	0.012	0.012	0.015	0.020	0.020	0.023	0.011	0.002	0.015	0.020	0.020	0.014	0.002	0.011

Table 3.

Kimura two-parameter pairwise genetic distances between populations of .

	009	011	012	024	047	048	049	050	051	052	053	054	055	056	057	058	059	060	062	064	068	069	070	071	072	073	074	134
009
011	0.000
012	0.000	0.000
024	0.002	0.002	0.002
047	0.000	0.000	0.000	0.002
048	0.000	0.000	0.000	0.002	0.000
049	0.002	0.002	0.002	0.003	0.002	0.002
050	0.000	0.000	0.000	0.002	0.000	0.000	0.002
051	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000
052	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000
053	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000
054	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000
055	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000
056	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000
057	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000
058	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
059	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
060	0.002	0.002	0.002	0.003	0.002	0.002	0.003	0.002	0.002	0.002	0.002	0.002	0.002	0.002	0.002	0.002	0.002
062	0.002	0.002	0.002	0.003	0.002	0.002	0.000	0.002	0.002	0.002	0.002	0.002	0.002	0.002	0.002	0.002	0.002	0.003
064	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002
068	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000
069	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000	0.000
070	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000	0.000	0.000
071	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000	0.000	0.000	0.000
072	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000	0.000	0.000	0.000	0.000
073	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000	0.000	0.000	0.000	0.000	0.000
074	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000
134	0.000	0.000	0.000	0.002	0.000	0.000	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.002	0.002	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000

Regarding the subgeneric concept of , our results (Figures 7, 8) do not support the traditional grouping except for , the selected species of which appear in the same clade. Our results rather support the monophyly of those species groups of Puthz (2008: 139–148), which we tested with at least two representatives. These species groups and the included species are: group with and , group with and , group with , and , group with , , and , group with , and .

Discussion

With the example of Iranian populations of the open-living and the stratobiont , we demonstrate that different ecomorphological forms of congeneric species with differing dispersal ability and degree of geneflow can show a different degree of infraspecific genetic variability.

The open-living is the most widespread in Iran. It was found in most of the country in high abundance at elevations between 250 m and 2800 m a.s. l. (Figure 5, after Serri and Frisch 2016: 27). As an example of the open-living ecomorphological form described by Kastcheev and Puthz (2011: 454), this mobile species does not show geographically structured populations. The low level of haplotype diversity as well as the low intraspecific distance of this species indicate a high level of gene flow between the populations of this species, which are connected to each other even across zoogeographic barriers due to the species’ dispersal ability. This gene flow within the Iranian meta-population of is probably supported by the wide ecological adaptability, which prevents geographic isolation. Unlike the remainder of Iranian , we repeatedly collected not only PageBreakin natural habitats, but also in polluted sites and anthropogenic places such as watering channels of farms far from natural, permanent watercourses.

Figure 5.

Distribution of in Iran (after Serri and Frisch 2016: 28).

The stratobiont , the second widespread in Iran, was collected in high abundance in most of the collecting sites all over the country (Figure 6, after Serri and Frisch 2016: 27). Our cladogram shows the separation of the tested populations into six genetic units, which can be explained by the limited dispersal ability of the mostly micropterous individuals of . The genetic variability of , as shown by the higher genetic distance among populations and more diverse haplotypes, might moreover be increased by discontinuity of suitable habitats caused by man-made destruction, because – in contrast to – the species usually avoids strongly disturbed sites.

Figure 6.

Distribution of in Iran (after Serri and Frisch 2016: 28).

Though our COI examination of a limited number of West Palaearctic species of is not extensive when it comes to understanding the supraspecific phylogeny of the entire clade, it clearly shows the monophyly of the included species and the polyphyletic relationship among the investigated members of subgenus . The relationships of and with other species were not resolved, because we were able to extract DNA from only one species of each of these subgenera. The monophyly of the selected species is, however, consistent with the result of the analysis performed by Koerner et al. (2013: 340).

Our results, which agree with those of Koerner et al. (2013: 345) and Lang et al. (2015: 20–21), further support the monophyly of the tested infrageneric species groups proposed by Puthz (2008: 139–148). On one hand, this result is not very significant, as only few species of some of these groupings were included in this study. On the other hand, our results clearly contradict the traditional subgeneric concept, which is followed until today, and proves the morphological characters this erroneous concept is based on to be phylogenetically uninformative convergencies. The included members of one of these traditional subgenera, , constitute, however, one well supported clade (bootstrap value >90) comprising the group, the group and the group. Particularly the first two species groups are closely related sister groups (bootstrap value 100). Further investigations are necessary to show whether – unlike the other traditional subgenera - actually represents a monophyletic group or not.

Our results support the supraspecific phylogenetic concept of Puthz (2008: 139–148) and at the same time largely contradict the traditional subgenera. Therefore, these subgenera should not be used anymore in favour of the informal species groups, though the monophyly of some of them still has to be proved.

Among the collected specimens of , there are specimens which show differences in the structure of the median lobe of the aedeagus and in the spermatheca. The cladogram shows that these specimens form a separate clade although they have no geographic separation. Both morphological and genetic examination of a broader basis of specimens is necessary to clarify whether this form should be considered as a distinct species.

Since we did not succeed in extracting DNA from a large number of the recently collected species or from the Iranian material in Scheerpeltz solution collected by Senglet, it was not possible to include all Iranian species into the analysis. Moreover, the paucity of fresh specimens of many rare species did not allow us to use genetic data of these species in our phylogenetic analysis. Nevertheless, this preliminary study provides benchmark data for future phylogenetic investigations that include a higher number of taxa at a wider geographic scale and additional genes. Our current analysis based on a COI fragment suggests that the ‘barcoding fragment’ studied here can also be used for testing the phylogenetic validity of supraspecific groups.