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

Abstract 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 Stenus 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.
Stenus 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(Betz : 1097. The labial features are involved in catching prey in a sudden manner despite the limited reaction ability of the beetle (Betz 1999(Betz : 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 With about 2674 species (Puthz, unpublished), Stenus is one of the species-richest genera of animals in the world (Puthz 2012: 286).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 Stenus, two major ecomorphological forms can be distinguished, which Kastche ev 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 Scopaeina Mulsant and Rey, 1878(Frisch et al. 2002. 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 Stenus species were recorded (Serri and Frisch 2016: 18), among which S. erythrocnemus Eppelsheim, 1884 and S. callidus 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), S. erythrocnemus is an open-living species and S. callidus 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.  Stenus was traditionally divided into subgenera according to morphological characters. Based on European species only, Rey (1884: 31) introduced the six subgenera Hemistenus, Hypostenus, Mesostenus, Nestus, Stenus, and Tesnus. Later, Heyden (1905: 262) replaced Mesostenus with Parastenus because of a homonymy with a genus in the Hymenoptera. Ádám (1987: 135), however, synonymized Parastenus with the older name Hemistenus Motschulsky, 1860, because the type species of both subgenera are considered as subjective synonyms. Therefore he introduced the new subgenus Metastenus for a distinct species group of Hemistenus (Herman 2001(Herman : 2041, but later he (Ádám 2001: 126) replaced this name with Metatesnus because of primary homonymy with Metastenus Walker, 1834 in the Hymenoptera. According to Puthz (2009: 47), the genus group name Adamostenus Özdikmen & Darılmaz, 2008, an unnecessary replacement name for Metatesnus, is a junior synonym of Metatesnus. Puthz (2001: 35) also synonymized Nestus with Stenus 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 Coleoptera, Schülke and Smetana (2015: 802-847) still divided this genus into five subgenera, which are Hemistenus Motschulsky, 1860, Hypostenus Rey, 1884, Stenus Latreille, 1797, Metatesnus Ádám, 2001and Tesnus Rey, 1884. Puthz (2008 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 Steninae 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 Dianous Leach, 1819, the second genus of the Steninae, actually constitute a monophyletic group within Stenus. 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 Stenus 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 Stenus 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 polymerase chain reaction (PCR) was used to amplify a 5' end fragment of the mitochondrial cytochrome c oxidase subunit I (COI) 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  (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 Stenus callidus and S. erythrocnemus 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).

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 Stenus. The alignment was blasted against GenBank sequences and found to match with existing records of Stenus. 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 Maximum Parsimony (MP) 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 Nearest-Neighbor-Interchange (NNI) method, gaps treatment using all sites, the neighbor-joining (NJ) 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 Stenus erythrocnemus and more pronounced heterogeny in S. callidus. The selected populations of S. callidus 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 S. callidus from Kordestan Province show a low similarity of the COI gene and appear in different clades of the cladogram (Figures 7, 8).
The haplotype networks for COI of S. callidus and S. erythrocnemus (Figures 3, 4) comprise fourteen and four haplotypes, respectively. Haplotype diversity (h) was estimated at 0.911±0.034 for S. callidus and 0.267±0.107 for S. erythrocnemus. The nucleotide diversity (π i) of each species was calculated as 0.01348±0.00074 for S. callidus and 0.00045±0.00019 Table 2. Kimura two-parameter pairwise genetic distances between populations of Stenus callidus.  for S. erythrocnemus (Table 4). In S. callidus, no haplotype has an outstandingly high frequency, while S. erythrocnemus has a dominant haplotype (Hap_1) found in populations of the Elburz and Zagros Mountains and the central mountain ranges.
The maximum genetic distance among populations does not exceed 0.003% in S. erythrocnemus and is much higher in S. callidus with 0.028% (Tables 2, 3). The highest genetic distance as well as the highest haplotype diversity in the populations of S. callidus was observed in the central zone of the Zagros Mountains. In S. erythrocnemus, the highest genetic distance is among the populations of northwestern Iran.
Regarding the subgeneric concept of Stenus, our results (Figures 7, 8) do not support the traditional grouping except for Hemistenus, 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: S. guttula group with S. erythrocnemus and S. maculiger,S. cordatus group with S. araxis and S. turk,S. glacialis group with S. medus,S. persicus and S. schah,S. ochropus,S. pieperi and S. ressli,S. ater group with S. ater,S. hypoproditor and S. intricatus zoufali. 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 4). Sites with more than one haplotype number indicate several geographically close localities.

Discussion
With the example of Iranian populations of the open-living Stenus erythrocnemus and the stratobiont S. callidus, 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 S. erythrocnemus is the most widespread Stenus 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 openliving 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 S. erythrocnemus is probably supported by the wide ecological adaptability, which prevents geographic isolation. Unlike the remainder of Iranian Stenus, we repeatedly collected S. erythrocnemus not only 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, Hemistenus, constitute, however, one well supported clade (bootstrap value >90) comprising the S. cordatus group, the S. glacialis group and the S. ochropus-ludyi-coarcticollis group. Particularly the first two species groups are closely related sister groups (bootstrap value 100). Further investigations are necessary to show whether Hemistenus -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 S. araxis, 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.