Chemoecology (2015) 25:11–24 DOI 10.1007/s00049-014-0171-4 CHEMOECOLOGY

RESEARCH PAPER

Phylogenetic relationships and chemical evolution of the genera and (Coleoptera: Staphylinidae)

Carolin Lang • Lars Koerner • Oliver Betz • Volker Puthz • Konrad Dettner

Received: 17 April 2014 / Accepted: 23 September 2014 / Published online: 8 October 2014 Ó Springer Basel 2014

Abstract The subfamily , composed of the species. Our investigations based on two algorithms such genera Dianous Leach 1819 and Stenus Latreille 1797, as Maximum Likelihood and Bayesian analyses support the belongs to the family of staphylinid (Staphylini- chemotaxonomic approach. Furthermore, our results dae). Some unique features characterize Stenus beetles, clearly support former analyses concerning the evolution- e.g., a distinct prey-capture apparatus (not found in the ary origin of Dianous within Stenus, which suggests a genus Dianous) and special pygidial gland secretion con- secondary loss of the specialized prey-capture apparatus. stituents such as the alkaloids stenusine (1), norstenusine Finally, phylogenetic aspects based on the morphology of (2), 3-(2-methyl-1-butenyl)pyridine (3), cicindeloine (4)as Steninae are discussed. well as several terpenes like a-pinene (5), 1,8-cineole (6) and 6-methyl-5-heptene-2-one (7); (only 1, 2 and terpenes Keywords Steninae Chemotaxonomy found in Dianous). As the secretion composition of Stenus Molecular phylogeny COI 16S rRNA Histone H3 beetles is species specific, it can be used for a chemotax- onomic approach to investigate relationships within the Steninae. Based on the alkaloid gland content, Steninae can Introduction be grouped into three clusters: the piperidine-, the pyridine- and the epoxypiperideine group. To clarify the phyloge- The genera Stenus Latreille 1797 and Dianous Leach 1819 netic relationships within Stenus and between Stenus and (the only members of the subfamily Steninae) belong to the Dianous, and to evaluate our chemotaxonomic approach, large family of Staphylinidae (rove beetles). The we analyzed a combined dataset of three gene sequences genus Stenus is one of the most species-rich genera within aligned of mitochondrial cytochrome c oxidase I (COI), the kingdom. To date, 2,612 species and eight fossil 16S rRNA and Histone H3 of 17 Stenus and 4 Dianous species are known worldwide (Puthz unpubl.). The genus Dianous covers more than 223 species (Puthz unpubl.) with a main distribution range in Asia (India, China and Handling Editor: Michael Heethoff. Southeast Asia). Generally, all staphylinid beetles including Stenus and C. Lang (&) K. Dettner Department of Animal Ecology II, University of Bayreuth, Dianous are characterized by a slender habitus and short Universita¨tsstr. 30, 95440 Bayreuth, Germany elytra. Because of the freely movable and, therefore, more e-mail: [email protected] vulnerable abdomen, most staphylinid beetles employ a highly evolved defensive gland system (Dettner 1991, L. Koerner O. Betz Department of Evolutionary Biology of Invertebrates, Institute 1993). Wide ranges of chemical gland constituents are used for Evolution and Ecology, University of Tu¨bingen, Auf der for defense (Dettner 1987). The representatives of the Morgenstelle 28E, 72076 Tu¨bingen, Germany Steninae are also equipped with a multifunctional pygidial gland secretion that acts as a defense against predators V. Puthz Burgmuseum Schlitz, Naturwissenschaftliche Abteilung, (Dettner 1987). Other functions of the secretion are Vorderburg 1, 36110 Schlitz, Germany avoidance of colonization by microorganisms when 123 12 C. Lang et al.

Fig. 1 Pygidial gland secretion compounds of Stenus relevant for this study (Schildknecht 1970; Schildknecht et al. 1975; Kohler 1979; Neumann 1993; Lusebrink et al. 2009;Mu¨ller et al. 2012;): stenusine (1), norstenusine (2), 3-(2-methyl-1- butenyl)pyridine (3), cicindeloine (4), a-pinene (5), 1,8-cineole (6) and 6-methyl-5- heptene-2-one (7)

secretion is applied on the body surface (‘‘secretion The genus Dianous, previously regarded as a sister grooming’’, Kovac¸ and Maschwitz 1990; Betz 1999; genus to Stenus, is currently grouped into species groups I Lusebrink 2007; Lusebrink et al. 2008b) and an extraor- and II based on the morphology of the frons (Puthz 1981, dinary movement on the water surface called ‘‘skimming’’ 2000, 2005; Shi and Zhou 2011; Tang et al. 2011). Mem- (Piffard 1901; Billard and Bruyant 1905; Jenkins 1960; bers of Dianous group I are characterized by large eyes Schildknecht et al. 1975). The multifunctional pygidial similar to those of Stenus (Puthz 1981). Therefore, these gland secretion of the Steninae consists of various piperi- beetles were traditionally placed in the genus Stenus, until dine and pyridine-derived alkaloids as well as several it was recognized that they do not possess their typical terpenes (Fig. 1; Schildknecht 1970; Schildknecht et al. prey-capture apparatus (Puthz 1981). 1975; Kohler 1979; Lusebrink et al. 2009;Mu¨ller et al. However, not only morphological characters were used 2012). to clarify complicated phylogenetic relationships within In the past, the genus Stenus was grouped into subgenera this huge subfamily of the Steninae. As already mentioned, based on various morphological features by staphylinid the pygidial secretion of the Steninae is composed of specialists (Rey, Casey, Motschulsky in Hermann 2001; several constituents (Figs. 1, 2). The secretion composition Lusebrink 2007; Puthz 2008). Originally, the genus was is species specific (Lusebrink 2007; Schierling et al. 2013) grouped into six subgenera, Stenus, Nestus, Tesnus, He- and can be used for a chemotaxonomic approach based on mistenus, Hypostenus and Parastenus (see also the the distribution of gland compounds (Fig. 2; Schierling determination key of Lohse 1964, which uses a today et al. 2013). The pygidial gland system of Steninae consists outdated subgenus-concept) mainly based on morphologi- of a pair of small and large reservoirs with the corre- cal features. These characteristics, for example, are the sponding gland tissues (Lang et al. 2012; Schierling and appearance of the 4th segment of the metatarsi (simple or Dettner 2013). In the large reservoirs, the alkaloids stenu- bi-lobed), relative length of the 1st and 5th segments of the sine (1), norstenusine (2), 3-(2-methyl-1-butenyl)pyridine metatarsi, relative length of the metatarsi and metatibiae (3) and cicindeloine (4) are stored, whereas the small res- and presence or absence of abdominal paratergites (Cam- ervoirs contain the terpenes a-pinene (5), 1,8-cineole (6) eron 1930; Lohse 1964; Zhao and Zhou 2004; Koerner and 6-methyl-5-heptene-2-one (7). According to Schierling et al. 2013). Later, subgenera were taxonomically revised et al. 2013, most of the Central European Stenus species resulting in five valid subgenera: Stenus s.str., Hemistenus can be grouped into three main groups based on their Motschulsky 1860, Hypostenus Rey 1884, Metatesnus pygidial gland content, especially concerning the alkaloids Adam 1987 and Tesnus Rey 1884 (Puthz 2001, 2008). In 1, 2, 3, and 4. Most Steninae belong to the piperidine this revision, Nestus belongs to Stenus s. str., Hemistenus is group. The secretion of this group is characterized by redefined to Metatesnus and Parastenus is renamed to alkaloids 1 as main and 2 as secondary component. In Hemistenus. However, recent findings indicate that this addition, terpenes 5, 6 and 7 represent minor and trace classification seems artificial and does probably not reflect components in the secretion. The piperidine group is authentic phylogenetic relationships. Currently, the genus regarded as chemotaxonomically basal (Schierling et al. is grouped into a large number of monophyletic species 2013). Furthermore, the pygidial gland system of basal or groups based on a wide range of morphological characters primitive Steninae is characterized by the presence of well- (Table 1; Puthz 2008). distinct small reservoirs storing terpenes. This especially

123 Phylogenetic relationships and chemical evolution 13

Table 1 Stenus, Dianous and Euaesthetus species used for phylogenetic analyses

Genus Subgenus Species Species Location GenBank accession no. group/species complex Gene sequences COI Histone 16SrRNA H3 Ingroup Stenus Stenus ater juno Germany, JQ085760a KJ127165 KJ144853 s.str. (Paykull,1789) Schleswig- Holstein, Kiel

Canada, Alberta JQ085761a KJ127166 KJ144854

clavicornis clavicornis Germany, Baden- JQ085764a KJ127156 KJ144844 (Scopoli, Württemberg, 1763) Tübingen Germany, JQ085765a KJ127157 KJ144845 Schleswig- Holstein, Kiel comma comma Germany, Baden- JQ085769a KJ127158 KJ144846 (LeConte, Württemberg, 1863) Tübingen Canada, Alberta JQ085771a KJ127159 KJ144847

biguttatus Germany, JQ085772a KJ127146 KJ144834 (Linnaeus, Schleswig- 1758) Holstein, Strande Germany, JQ085773a KJ127147 KJ144835 Schleswig- Holstein, Strande boops boops Germany, Baden- JQ085778a KJ127150 KJ144838 (Ljungh, Württemberg, 1804) Tübingen Germany, Baden- JQ085779a KJ127151 KJ144839 Württemberg, Tübingen canaliculatus canaliculatus Germany, Baden- JQ085780a KJ127154 KJ144842 (Gyllenhal, Württemberg, 1827) Tübingen Germany, Baden- JQ085781a KJ127155 KJ144843 Württemberg, Tübingen

nitens Germany, Baden- JQ085782a KJ127169 KJ144857 (Stephens, Württemberg, 1833) Tübingen humilis humilis Germany, Baden- JQ085783a KJ127160 KJ144848 (Erichson, Württemberg, 1839) Tübingen Germany, Baden- JQ085784a KJ127161 KJ144849 Württemberg, Tübingen Tesnus brunnipes brunnipes Germany, Bavaria, KJ509590 KJ127152 KJ144840 (Stephens, Fichtelgebirge, 1833) Gemös Germany, Bavaria, KJ509591 KJ127153 KJ144841 Fichtelgebirge, Gemös crassus intermediusb Germany, KJ509592 KJ127163 KJ144851 (Rey, 1884) Mecklenburg- Vorpommern, Insel Poel Germany, KJ509593 KJ127164 KJ144852 Mecklenburg- Vorpommern, Insel Poel Hypostenus similis similis Germany, Bavaria, JQ085787a KJ127170 KJ144858 (Herbst, 1784) Creußen solutus Germany, JQ085789a KJ127171 KJ144859

123 14 C. Lang et al.

Table 1 continued

Erichson Schleswig- 1840) Holstein, Strande Germany, JQ085790a KJ127172 KJ144860 Schleswig- Holstein, Strande tarsalis tarsalis Germany, JQ085794a KJ127173 KJ144861 (Ljungh, Schleswig- 1804) Holstein, Flintbek Germany, Bavaria, JQ085795a KJ127174 KJ144862 Limmersdorfer Forst Metatesnus bifoveolatus bifoveolatus Germany, JQ085796a KJ127145 KJ144833 (Gyllenhal, Schleswig- 1827) Holstein, Kiel binotatus binotatus Germany, JQ085798a KJ127148 KJ144836 (Ljungh, Schleswig- 1804) Holstein, Flintbek Germany, JQ085799a KJ127149 KJ144837 Brandenburg, Glienicke not defined yet latifrons Germany, JQ085792a KJ127167 KJ144855 (Erichson, Schleswig- 1839) Holstein, Flintbek Germany, JQ085791a KJ127168 KJ144856 Schleswig- Holstein, Flintbek Hemistenus impressus impressus Germany, JQ085804a KJ127162 KJ144850 (Germar, Schleswig- 1824) Holstein, Kiel Dianous Group I fengtingae (sp. China, Hainan KJ509585 KJ127140 KJ144828 n.) Prov., Ledong County, Jianfengling Group II Coerulescens coerulescens Germany, Bavaria, JQ085805a KJ127138 KJ144826 (Gyllenhal, Röslau, Eger Falls 18109 Aereus- vietnamensis China, Hainan KJ509586 KJ127141 KJ144829 andrewesi (Puthz, 1980) Prov., Ledong County, Jianfengling Ocellatus fellowesi China, Hainan KJ509584 KJ127139 KJ144827 (Puthz, 2005) Prov., Wuzhishan City, Mt. Wuzhishan Outgroup ruficapillus Germany, Bavaria, KJ509587 KJ127142 KJ144830 Euaesthetus (Lacordaire, Lichtenfels, Main- (Euaesthetinae) 1835) überschwemmungs- geniste Germany, Bavaria, KJ509588 KJ127143 KJ144831 Lichtenfels, Main- überschwemmungs- geniste Germany, Bavaria, KJ509589 KJ127144 KJ144832 Lichtenfels, Main- überschwemmungs- geniste

Species are grouped according to genus, species complexes (Dianous; Puthz 1981, 2000, 2005; Shi and Zhou 2011; Tang et al. 2011)/species groups (Stenus; Puthz 2006, 2008) and subgenus (see also Koerner et al. 2013). Members of chemotaxonomic groups according to Schierling et al. 2013 are highlighted in color; red piperidine group, yellow pyridine group, blue epoxypiperideine group; members of Dianous complex I and II (Puthz 1981, 2000, 2005; Shi and Zhou 2011; Tang et al. 2011) are marked in green a Sequences taken from GenBank b Beetles obtained from Dr. Alexander Kleeberg, Berlin

123 Phylogenetic relationships and chemical evolution 15

Fig. 2 Chemotaxonomic approach introduced by Schierling et al. eversible labium with distal adhesive prey-capture apparatus (Puthz 2013; modified. Piperidine group is the most extensive group, species 1981; Leschen and Newton 2003) opened circle short, barely listed there are only examples. Chemotaxonomic groups are high- eversible labium missing an adhesive prey-capture apparatus 6— lighted in color; red piperidine group (primitive), yellow pyridine closed circle only tip of inner paramere surface bristled (Puthz 1981) group (derived), blue: epoxypiperideine group (most derived). Clade opened circle whole inner paramere surface bristled 7—closed circle supported by (Schierling et al. 2013): 1—closed circle molecular data extension of the alkaloid repertoire by 3-(2-methyl-1-butenyl)pyridine (18S rDNA; Grebennikov and Newton 2009) 2—closed circle 18S (3), decrease in norstenusine (2) and terpene (5–7) opened circle only rDNA, bootstrapping results 79–97 % (Grebennikov and Newton stenusine (1) and norstenusine (2) available, terpenes (5–7) generally 2009) 3—closed circle 18S rDNA, bootstrapping results 99–100 % present significantly 8—closed circle extension of the alkaloid (Grebennikov and Newton 2009) 4—closed circle invagination of the repertoire by cicindeloine (4); stenusine (1) and norstenusine (2) pleural membrane between 9. and 10. abdominal segment forming available only in minimal traces opened circle only 3-(2-methyl-1- paired komplex glands (Naomi 1985); biosynthesis of defense butenyl)pyridine (3), stenusine (1) and traces of norstenusine (2) alkaloids stenusine (1) and norstenusine (2); opened circle komplex present; 3-(2-methyl-1- butenyl)pyridine (3) and stenusine (1) as main glands at abdominal segments 9 and 10 absent 5—closed circle long constituents applies to S. comma and S. biguttatus. The presence of In summary, different approaches have been used to secretion composition of 1, 2, 5 and 6 indicates that Dia- clarify the phylogenetic relationships within genus Stenus nous coerulescens, the only Central European species of and between Stenus and Dianous. Some studies deal with Dianous, is a member of the piperidine group (Schierling the position of the Steninae within staphylinid beetles, e.g., et al. 2013). The next more derived taxonomic level is Puthz (1981), Hansen (1997), Leschen and Newton (2003), represented by the pyridine group. Species of this group Thayer (2005), Grebennikov and Newton (2009) and exhibit 1 and especially 3 as main components and 2 as Clarke and Grebennikov (2009). According to Grebenni- secondary component in their secretion. The amount of kov and Newton (2009), the monophyly of Steninae is terpenes fraction is greatly reduced, as well as the size of supported by many larval and adult autapomorphies and small reservoirs and their gland tissues (Schierling et al. suggested also by molecular analysis. There are only a few 2013). Species like S. similis and S. tarsalis belong to the molecular analyses within genus Stenus. Only one study pyridine group. The most evolved taxonomic level is discusses associations within the Steninae including described by epoxypiperideine group with only a few assumptions regarding the position of Dianous (Koerner members represented by S. binotatus and S. solutus in this et al. 2013). This study revealed Dianous clustering within study. The gland secretion repertoire of this group is a paraphyletic Stenus, indicating a secondary loss of the extended to alkaloid constituent 4 as main component, in prey-capture apparatus. Their study was based on molec- which 3 serves as secondary component at a high con- ular investigations only using sequences of the centration, and constituents 1 and 2 only as trace mitochondrial cytochrome c oxidase I (COI). components. The species of this group completely lack The aim of our study was to investigate the chemical terpenes like 5, 6 and 7 and, therefore, also the primitive evolution within genus Stenus by means of molecular small reservoirs (Schierling et al. 2013). phylogenetic methods firstly using a combination of three

123 16 C. Lang et al. gene sequences (mitochondrial cytochrome c oxidase I Primers, PCR and sequencing (COI), 16S rRNA, Histone H3) instead of implementing solely the sequences of COI in phylogenetic analyses. In For extraction of total genomic DNA from beetle tissue, our discussion, some important minor aspects like species the High Pure PCR Template Preparation Kit (Roche groups and species complexes introduced by Puthz (1981, Applied Science, Mannheim, Germany) was used. The 2000, 2005, 2006, 2008), Shi and Zhou (2011) and Tang extraction procedure followed the manufacturer’s guide- et al. (2011) are considered, as well as a support of the lines. The Polymerase chain reaction (PCR) was conducted position of Dianous within a paraphyletic Stenus (Koerner with GoTaqÒ Hot Start Polymerase (Promega, Madison, et al. 2013). However, the focus of our study is the USA). The total reaction volume of 25 ll contained chemical evolution of Steninae with a discussion based on 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 mM of each the chemotaxonomic approach of Schierling et al. 2013 primer and 1.25 Units of GoTaqÒ DNA polymerase. PCR (Fig. 2). Our results maintain the assumptions of Schierling conditions were: an initial denaturation step at 95 °C for et al. (2013) and demonstrate a possible process of evolu- 2 min, followed by 9 cycles of 95 °C (30 s) denaturation, tion regarding chemical gland constituents of Steninae. 46 °C (1 min) annealing and 72 °C (45 s) extension and subsequently 34 cycles of 95 °C (30 s) denaturation, 51 °C (1 min) annealing and 72 °C (45 s). The PCR was termi- Materials and methods nated at 72 °C (5 min) for final extension (Koerner et al. 2013). For amplification of desired sequence fragments Examined beetle material and preparation for DNA during the PCR, different corresponding primer sets were isolation used for mitochondrial cytochrome c oxidase I (COI), 16S ribosomal RNA and Histone H3 gene (Table 2). Since it Molecular analyses were conducted with 17 species of Stenus, was not possible to gain the 1200 bp fragment (mtD4 and 4representativesofDianous and 3 specimens of Euaesthetus Pat; Table 2) of COI from all specimens during PCR due to ruficapillus as an outgroup (Table 1). Beetles collected in difficulties, it was necessary to apply a second forward Germany were identified using the key of Lohse (1964), primer for COI (800 bp; Jerry and Pat). For some ampli- additions of Lohse (1989) and the new identification key of fication reactions concerning 16S ribosomal RNA, we Puthz (2001, 2008). When possible, two examples per species designed a new pair of primers called 16S_Stenus and from different localities were analyzed. Before identification, purchased those from Eurofins MWG Operon, Ebersberg, the beetles were killed by freezing; afterwards, the Germany. were stored at -20 °C until further preparation. During dis- After amplification, PCR products gained were purified section, the abdomen and entire digestive system were using the Thermo Scientific GeneJET PCR Purification Kit removed to prevent contamination of beetle DNA by foreign (Thermoscientific, Schwerte, Germany). Purification was DNA of prey animals or parasites (Maus et al. 2001). Until performed on a silica membrane. Purification procedure DNA isolation, specimens were kept at -20 °C. The authors has been performed following the manufacturer’s protocol. collected most beetles. Dr. Alexander Kleeberg, Berlin, GATC Biotech, Ko¨ln, Germany, conducted sequencing kindly provided additional specimens. COI sequences of 14 reaction of the purified PCR products in both directions. Stenus and 2 Dianous species were taken from GenBank All sequences were submitted to GenBank and accession (Koerner et al. 2013, Table 1). numbers were received (Table 1; www.ncbi.nlm.nih.gov).

Table 2 Primers used and length of amplified sequence fragments Gene sequence Primer sequence Citation Fragment length (bp)

COI Jerry_F fwd 50-CAA CAT TTA TTT TGA TTT TTT GG-30 Simon et al. 1994 (Jerry-Pat) 800 mtD4 fwd 50-TAC AAT TTA TCG CCT AAA CTT CAG CC-30 Sperling and Hickey 1994 1200 (mtD4-Pat) Pat_R rev 50-TCC AAT GCA CTA ATC TGC CAT ATT A-30 16S rRNA 16S_Fneu fwd 50-CGC CTG TTT ATC AAA AAC AT-30 Kocher et al. 1989 650 16S_Rneu rev 50-CCG GTC TGA ACT CAG ATC A-30 16S_Stenus_F fwd 50-CGC CTG TTT ATC AAA AAC AT-30 600 16S_Stenus_R rev 50-TTA ATC CAA CAT CGA GGT C-30 Histone H3 H3_F fwd 50-ATG GCT CGT ACC AAG CAG ACG GC-30 Colgan et al. 1998 300 H3_R rev 50-TCC TTG GGC ATG ATG GTG AC-30

123 Phylogenetic relationships and chemical evolution 17

Sequence analysis and phylogeny different sequence evolution models were applied and heuristic tree search strategies as well as bootstrapping Sequences of the three genes were aligned separately using procedure were the same as mentioned above. the ClustalW algorithm (Thompson et al. 1994) imple- In addition to phylogenetic analyses performed with mented in BioEdit 7.1.3.0 (Hall 1999) and the Muscle PAUP, Bayesian phylogeny estimation (BA) with MrBayes algorithm (Edgar 2004) implemented in MEGA 5.2.2 3.2.2 (Ronquist and Huelsenbeck 2003) was conducted as (Tamura et al. 2011). In addition, alignments were well. First, the three genes were analyzed separately in improved manually and ambiguous positions were consideration of sequence evolution model parameters. For removed. Afterwards, best-fitting models of sequence evo- aligned matrices of Histone H3- and 16S rRNA sequences, lution were estimated for aligned sequence matrices using two runs with four simultaneous Markov chains in three Aikaike Information Criterion (AIC) and Maximum Like- million generations were performed to reach a standard lihood (ML) optimized parameters implemented in deviation of split frequencies below 0.01; for COI two runs jModelTest2 (Posada 2008; Guindon and Gascuel 2003; with two million generations were adequate. In all analy- Darriba et al. 2012). Resulting models and corresponding ses, obtained trees were sampled every 1000 parameters are summarized in Table 3. For phylogenetic (samplefreq = 1000) and the burn-in was determined analyses based on Maximum Likelihood algorithm, PAUP* appropriately (regarded as burn-in for COI, Histone H3 and 4.0 b10 has been used (Swofford 1998). The analyses were 16S rRNA: first 70 trees, first 200 trees and first 65 trees, performed using the starting tree gained from stepwise respectively). For the tree containing sequences of all three addition and 100 replicates of random order. For heuristic genes analyzed, formerly described combined character set tree search, the tree-bisection–reconnection (TBR) algo- of sequences of COI, 16S rRNA and Histone H3 was rithm for branch swapping was applied which provided partitioned again and model parameters applied to corre- best-supported clades. Reliability of nodes was ascertained sponding partitions. Bayesian analyses were performed from 100 bootstrap replicates with ML parameters and NJ with three runs with four simultaneous Markov chains in as tree-reconstruction method for ML. Furthermore, branch one million generations. After three million generations, lengths were saved. This procedure was the same for single- standard deviation of split frequencies was below 0.01. gene sequence alignments of COI, 16S rRNA and Histone First 50 trees were regarded as burn-in and discarded. All H3. For strict consensus tree (Koerner et al. 2013) based on conducted Bayesian analyses were initiated using default the sequences of COI, 16S rRNA and Histone H3, a com- random tree option and branch lengths were saved. Fur- bined character set was used which was partitioned thermore, posterior probabilities were estimated for all according to corresponding genes and coding and non- analyses and trees were combined to 50 % majority rule coding sections (Hemp et al. 2010). The parameters of consensus trees.

Table 3 Parameters of sequence evolution models found by jModelTest2 (Posada 2008; Guindon and Gascuel 2003; Darriba et al. 2012) based on AIC; GTR general time reversible Gene Sequence Base Substitution Proportion of a-Shape Number of Number of sequence evolution frequencies rates invariable sites parameter substitution rate categories alignment model selected types

COI GTR?G?IA= 0.3144 A - C = 1.0000 0.4520 0.8040 6 4 C = 0.1126 A - G = 10.4582 G = 0.1302 A–T= 7.4249 T = 0.4428 C–G= 3.5269 G - T = 34.4309 16S rRNA GTR?GA= 0.3310 A - C = 3.4472 0 0.2330 6 4 C = 0.1276 A–G= 17.2541 G = 0.1632 A–T= 65.1478 T = 0.3782 C–G= 1.0000 G–T= 65.1478 Histone H3 GTR?G?IA= 0.2340 A–C= 1.0000 0.1790 0.2300 6 4 C = 0.2913 A–G= 4.9433 G = 0.2674 A–T= 0.4152 T = 0.2073 C–G= 3.0862 G–T= 4.9433

123 18 C. Lang et al.

Results To evaluate the position of chemotaxonomic group members within phylogenetic trees, two different algorithms Sequence features and statistic and analyses were used to assess and describe the phyloge- netic relationships within the genus Stenus and between the Alignments of COI, 16S rRNA and Histone H3 of each of genera Stenus and Dianous. Maximum Likelihood (ML)- the 37 sequences contained 22 species (1 outgroup, 1 Dia- and Bayesian Analyses (BA) trees were constructed and nous group I, 3 Dianous group II, 17 Stenus species. The compared containing only sequences of COI, 16S rRNA or aligned matrix of COI sequences consisted of 1203 posi- Histone H3, respectively. Because of strong inconsistencies tions, 391 of them being parsimony informative and 812 concerning the position of chemotaxonomic group members being variable. The alignment of 16S rRNA sequences within the trees, two consensus trees of ML and BA analyses consisted of 613 positions, 232 of them being parsimony were built. Solely all Dianous species clustered together informative and 381 being variable. The aligned matrix of within Stenus in all constructed trees based on single-gene Histone H3 consisted of 316 positions, 96 of them being sequence alignments. Only the consensus tree based on BA parsimony informative and 220 being variable. In addition, analyses is shown (Fig. 3), since both consensus trees of ML uncorrected p-distance values were calculated for different and BA analyses were characterized by almost the same groupings, e.g., Stenus and Dianous and chemotaxonomic topology, similar or identical clade support values and groups. Concerning the aligned matrix of COI sequences, furthermore contained no inconsistencies regarding che- highest uncorrected sequence p-distance within ingroup motaxonomic aspects. In both trees, consistent tree (Steninae) has been found to be 16.5 % between S. tarsalis topologies could be found with high BPP (Bayesian Pos- and S. latifrons. Regarding the alignment of 16S rRNA, terior Probability; BA) and Bootstrap value (ML) support. highest uncorrected sequence p-distance within ingroup All Dianous species (of both group I and II) formed a (Steninae) has been found to be 40.1 % between S. similis consistent cluster with counterpart position of group I and S. intermedius. Moreover, highest uncorrected member D. fengtingae towards other Dianous species D. sequence p-distance within ingroup (Steninae) has been coerulescens, D. fellowesi and D. vietnamensis of group II found to be 20.2 % between Canadian S. comma and S. (BPP = 1.00). Out of the Dianous species used in this study, canaliculatus concerning the aligned matrix of Histone H3 D. coerulescens is the only one, whose gland content has sequences. Further results and additional groupings of these been analyzed by GC/MS (Gas Chromatography–Mass calculations are summarized in Table 4. Spectrometry). D. coerulescens is characterized by a stenusine (1)-based secretion with a considerable amount of Phylogenetic analyses terpenes 5 and 6 (a-pinene and 1,8-cineole) in the small reservoirs (Schierling et al. 2013) and belongs to chemo- In this study, main focus of attention is given to chemotax- taxonomic basal piperidine group. All Dianous species onomic aspects introduced by Schierling et al. 2013 (Fig. 2) occupy a phylogenetic position in direct neighborhood to and the survey of these results on a molecular phylogenetic other basal piperidine group members of Stenus (e.g., S. basis. In addition, the position of Dianous clustering within clavicornis, S. juno and S. humilis). Most Stenus species of presumed paraphyletic Stenus (Koerner et al. 2013) and chemotaxonomic basal piperidine group cluster together. species groups based on morphological features introduced These group members form clusters with high BPP support by Puthz (2008) are treated as minor aspects. within the tree, e.g., S. clavicornis, S. juno and S. humilis

Table 4 Uncorrected Gene sequence p-distance values (%) calculated alignments for different sequence groupings Groupings COI 16S rRNA Histone H3

Uncorrected Within Stenus 12.4 15.0 12.7 p-distances (%) Within Dianous 10.9 8.2 8.8 Between Stenus- Dianous 13.3 13.0 14.0 Within piperidine group 12.7 15.5 13.2 Within pyridine group 9.6 7.0 9.6 Within epoxypiperideine group 8.7 6.5 10.9 Between piperidine–pyridine 13.8 14.0 12.9 Between piperidine–epoxypiperideine 13.1 14.1 12.7 Between pyridine–epoxypiperideine 12.9 9.0 9.0

123 Phylogenetic relationships and chemical evolution 19

Fig. 3 Maximum a posterior (MAP) tree resulting from Bayesian colored; red: piperidine group, yellow: pyridine group, blue: epox- analysis (Ronquist and Huelsenbeck 2003). The numbers above the ypiperideine group; members of Dianous complex I and II (Puthz branches indicate Bayesian Posterior Probabilities C0.50. Members of 1981, 2000, 2005; Shi and Zhou 2011; Tang et al. 2011) are marked chemotaxonomic groups according to Schierling et al. 2013 are in green; root with E. ruficapillus not shown

(BPP = 0.98), S. biguttatus and S. comma (BPP = 1.00), S. Based on chemotaxonomic aspects and pygidial gland boops, S. canaliculatus, S. nitens, S. intermedius and S. morphology, S. comma and S. biguttatus form a cluster and impressus (BPP = 0.95) and finally S. brunnipes and S. feature well-distinct small reservoirs with a high amount of latifrons (BPP = 0.63). In addition, S. intermedius shows terpenes (Schierling et al. 2013). Regarding the subgenus- the highest number of evolutionary changes resulting in long concept mentioned in the introduction section, piperidine branch lengths (Fig. 3). group members do not maintain this grouping as, e.g.,

123 20 C. Lang et al.

Tesnus species S. brunnipes forms a cluster with Hypost- et al. 2003; Ribera et al. 2003, 2004; Pons et al. 2006; Hunt enus member S. latifrons. Furthermore, S. impressus et al. 2007; Heethoff et al. 2011). On one hand, its universal (Hemistenus) can be found in a cluster together with S. primers are very robust; on the other hand, it possesses a intermedius (Tesnus), S. nitens, S. canaliculatus and S. greater range of phylogenetic signals than any other mito- boops (all former Nestus species; now Stenus s. str.; Puthz chondrial gene (Hebert et al. 2003). In addition to COI, a 2008). Only a few species groups defined by Puthz (2008) gene sequence coding for Histone H3 nucleosomal core (Table 1) are supported, like S. canaliculatus group con- protein was used in our study. This gene predominantly sisting of S. canaliculatus and S. nitens and in addition S. comprises highly conserved regions (Baldo et al. 1999; comma group comprising S. comma and S. biguttatus each Dinapoli et al. 2007; Thatcher and Gorovsky 1994) and is forming a small cluster in the BA-tree (both BPP = 1.00). especially suitable for phylogenetic analyses—particularly The next chemotaxonomic level is represented by pyr- on a generic level (Dinapoli et al. 2007). Investigations idine group members S. similis and S. tarsalis possessing were also conducted on 16S rRNA sequences forming the the chemotaxonomic-derived gland compound 3-(2- third gene used. This sequence is not only widely used methyl-1-butenyl)pyridine (3) in their pygidial glands particularly in bacterial phylogenetic analyses (‘‘house- (Schierling et al. 2013). Although these two species cannot keeping gene’’; Janda and Abbott 2007), but also possesses be found in the same clade, they are part of a major cluster important desirable features for invertebrate studies, such as comprising the species S. bifoveolatus, S. bintotatus and S. extreme conservation, presence in every cell with same solutus (BPP = 0.98). Interestingly, basal piperidine group function and a high number of copies per cell (Cilia et al. member S. bifoveolatus (Metatesnus) belongs to this clus- 1996; Doolittle 1999). Because consistent topologies ter, which contains chemotaxonomic-derived species. In appeared in both trees constructed depending on BA and this case, the subgenus-concept was also not maintained: ML analyses, selected gene sequences seem to be suitable to Hypostenus species (S. similis, S. tarsalis and S. solutus) resolve phylogenetic relationships within Steninae. How- cluster together with Metatesnus species (S. bifoveolatus ever, similar to the study of Maus et al. (2001) conducted on and S. binotatus). the staphylinid genus Aleochara, basal phylogenetic reso- The most evolved chemotaxonomic level is represented lution appears unsatisfying. A possible reason for the lack by the epoxypiperideine group comprising S. binotatus and of basal resolution may have been multiple divergence S. solutus in our study. Species of this group are charac- events over a short time frame (Maddison 1989; Koerner terized by extension of pygidial gland repertoire to the new et al. 2013). In addition, missing basal resolution may also alkaloid cicindeloine (4) as main gland constituent (Schi- have originated in a phase of rapid evolution in which erling et al. 2013). Alkaloids 1 and 2 are completely branching events occurred in a short time (Maus et al. replaced by compounds 3 and 4. In the tree constructed, S. 2001). Since our study is based on a few taxa and only three solutus is positioned in direct neighborhood of pyridine gene sequences, further investigations with additional taxa group member S. similis, which also possesses the chemo- and more gene sequences could contribute positively to a taxonomic-derived gland constituent 3, but lacks the most more exact basal resolution within the genus Stenus. evolved compound 4 in its glands. Concerning species groups of Puthz (2008), the S. similis group consisting of S. Partial support of morphological monophyletic groups similis and S. solutus (Table 1) is supported (BPP = 0.83). The other epoxypiperideine group member S. binotatus As mentioned before, several different approaches to occupies an isolated position regarding S. solutus within a classify the species-rich genus Stenus into several groups major cluster of both chemotaxonomic-derived and most exist, e.g., traditional grouping into subgenera (Cameron evolved species. However, all Stenus species possessing 1930; Lohse 1964; Zhao and Zhou 2004). This grouping chemotaxonomic evolved gland compounds show a close was based on special morphological features like mor- relationship within the tree constructed (Fig. 3). phology and length of metatarsi and metatibiae and appearance of abdominal paratergites. Our results do not support this grouping, since species with bi-lobed and Discussion simple metatarsi cluster together forming poly- or para- phyletic groups in both constructed trees (for example S. Gene selection latifrons owning bi-lobed metatarsi forms a group with simple metatarsi possessing S. brunnipes; Fig. 3). These Three different gene sequences were used for our phylo- findings are supported by analyses of Stenus based on COI genetic analyses of Steninae. The COI gene is often used as gene sequences conducted by Koerner et al. 2013. taxonomic standard barcode for both species identification Because of problems regarding the traditional subgenus and biogeographic analyses (Folmer et al. 1994; Hebert concept in Stenus, Puthz (2001, 2008) introduced a large 123 Phylogenetic relationships and chemical evolution 21 number of monophyletic species groups based on a wide in Dianous species and disproved former assumption of range of morphologic features, e.g., structure of the aede- Dianous being a more primitive precursor of Stenus. Our agus. Some of these monophyletic groups are supported by molecular phylogenetic study based on three gene our study, such as S. similis-, S. canaliculatus- and S. sequences of COI, 16S rRNA and Histone provides further comma group. However, only two representatives of each evidence that Dianous belongs to a paraphyletic Stenus of these groups were considered, whereas other groups are (Fig. 3). not supported. Further investigations with a higher number of species are necessary to identify these morphological Comparison with chemotaxonomic approach monophyletic groups on a molecular phylogeny basis. There are additional relevant results which in most cases Schierling et al. 2013 grouped most of the Central Euro- are in accordance with Puthz’ taxonomic opinion (Puthz pean Stenus species into three chemotaxonomic groups unpubl.). The species S. canaliculatus, S. nitens and S. depending on their pygidial gland content. Among all intermedius are members of the same cluster together with investigated staphylinid beetles occurrence of different S. boops (Fig. 3). Regarding the first three species men- alkaloids in pygidial glands is limited to the Steninae tioned above, this result appears plausible since these three (Schierling et al. 2013). Dianous coerulescens, represent- species are characterized by their sclerotized spermatheca. ing the only European Dianous species, possesses the same However, S. boops lacking a sclerotized spermatheca piperidine alkaloid constituents as Stenus: stenusine (1) and should not be positioned in direct neighborhood to the norstenusine (2) and, therefore, belongs to the basal other species stated. In addition, S. juno, S. clavicornis and piperidine group (Schierling et al. 2013). Since all inves- S. humilis forming a cluster possess the similar appearance tigated Dianous species cluster within Stenus in the direct of aedeagus. Finally, D. fengtingae belonging to Dianous neighborhood of piperidine group members (Fig. 3), our complex I (Puthz 1981, 2000, 2005; Shi and Zhou 2011; results are in accordance with findings of Schierling et al. Tang et al. 2011) serves as counterpart to Dianous species 2013. Recently, gland contents of some Asian Dianous of complex II represented in our study by D. coerulecens, species from Thailand, D. obliquenotatus, D. karen (both D. fellowesi and D. vietnamensis (Fig. 3). Future molecular Dianous group II) and D. betzi (Dianous group I) were phylogenetic investigations dealing with more representa- investigated with GC–MS. The pygidial glands only con- tives of Dianous complex I are needed to confirm its tain the chemotaxonomic basal alkaloids 1 and 2 (Lang neighboring position with respect to Dianous complex II. unpubl.). These results give further evidence of Dianous belonging to the basal piperidine group within Stenus. Affirmation of Dianous’ origin within Stenus Not only representatives of the basal piperidine group form clusters within both constructed trees (Fig. 3). In The prey-capture apparatus has always been regarded as a addition, members of both chemotaxonomic-derived alka- most prominent autapomorphic character of Stenus pro- loid groups show a special arrangement, too (Fig. 3). viding evidence of monophyly of this genus referring to Interestingly epoxypiperideine group representative S. bi- Dianous as ‘‘sister genus’’. This unique apparatus to catch notatus occupies an isolated position within a cluster elusive prey in a rapid and effective manner consists of a containing other Stenus species featured by derived alka- protruding elongated labium with paraglossae modified loid gland contents such as S. similis, S. tarsalis and S. into adhesive pads (Weinreich 1968; Puthz 1981;Betz solutus. To provide possible explanations for this special 1996, 2006; Koerner et al. 2013). Dianous beetles lack arrangement of S. binotatus, it is necessary to pay attention such a specialized labium; their labium is much shorter, to the stereochemistry of cicindeloine (4). Pygidial glands only slightly protrudable and lacks the adhesive pads of members of epoxypiperideine group S. solutus and S. (Weinreich 1968; Puthz 1981). The phylogenetic relation- cicindeloides (the latter was not included in our study) ship of Dianous and Stenus has already been discussed in contain (2S,3S,10S)-cicindeloine whereas S. binotatus pos- several studies (Puthz 1981; Clarke and Grebennikov sesses the (2S,3S,10R)-isomer of cicindeloine (Schierling 2009). In 2009, Grebennikov and Newton suggested three et al. 2012). To date, there is no knowledge about which autapomorphic characters for genus Dianous, but their gene sequence(s) is (are) involved in biosynthesis of 4. investigation was based solely on one Dianous species. Indeed gene sequences used in our study are not involved Based on COI sequences, Koerner et al. 2013 were the first in biosynthesis of gland constituent 4, but possession of to conduct molecular phylogenetic analyses of a variety of (2S,3S,10R)-cicindeloine could provide an indication for Dianous and many representatives of Stenus. This study general genetic differences of S. binotatus in comparison gives an indication of Dianous clustering into a paraphy- with other epoxypiperideine group representatives. letic Stenus. Consequently, Koerner et al. 2013 proposed a The chemotaxonomic approach of Schierling et al. 2013 secondary reduction of the distinct prey-capture apparatus is supported quite well by our phylogenetic analyses. 123 22 C. Lang et al.

Solely the position of epoxypiperideine group members S. cicindeloine was featured by the lowest spreading pres- binotatus and S. solutus not sharing exactly the same clade sure in comparison with other alkaloids 1, 2 and 3 (Lang seems problematic. This problem applies also to pyridine et al. 2012). Therefore, S. solutus is not able to skim as group members S. tarsalis and S. similis. However, a close rapidly as S. comma.However,S. solutus and S. comma molecular phylogenetic relationship of all investigated do not inhabit the same biotope; whereas skimming is chemotaxonomic-derived Stenus species can be assumed, significant for survival of S. comma in open biotopes since these beetles form the same cluster and possess the near waters, avoidance of infestation by microorganisms transition-compound 3-(2-methyl-1-butenyl)pyridine (3)in is significant for S. solutus living in marshes and wet their glands. detritus (Horion 1963)favoringgrowthofharmful However, the position of S. bifoveolatus belonging to microorganisms. Gland compounds 3 and 4 provide the basal piperidine group adjacent to chemotaxonomic- more effective protection against infestation of micro- derived species S. similis, S. tarsalis, S. solutus and S. bi- organisms than 1 and 2 when spread on the beetle’s body notatus (Fig. 3) cannot be explained by chemotaxonomy. surface (Kovac¸ and Maschwitz 1990;Betz1999;Luse- In this case, other attributes of the beetles need to be brink 2007; Lusebrink et al. 2008b;Schierlingetal. considered. 2013). It can be assumed that species-specific secretion Regarding alkaloid biosynthesis, another phylogenetic composition of every Stenus speciesisadjustedtohave hypothesis concerning the chemotaxonomic approach of the best-adapted composition concerning the character- Schierling et al. 2013 is possible. Steninae’s alkaloids 1, istics of the habitat the beetles live in. Therefore, 3 and 4 are synthesized based on amino acids L-lysine secondary reduction of derived alkaloids in basal piper- and L-isoleucine (Lusebrink et al. 2008a,b,Schierling idine group members is imaginable in process of et al. 2011, 2012). The amino acid L-lysine provides the chemical evolution. However, tree topology concerning N-heterocyclic ring whereas L-isoleucine forms the side representatives of both pyridine- and epoxypiperideine chain. The different alkaloids are finally obtained by group (Fig. 3) point to a close relationship between slight modifications of shared precursor molecules species of a higher chemotaxonomic evolved level based (Schierling et al. 2012). Because of this more or less on COI, 16S rRNA and Histone sequences. simple biosynthetic pathway and ubiquitous availability of required amino acids, it is easily possible for the beetles to synthesize all alkaloids occurring in every Stenus species. So why do glands of some species con- Conclusion tain no derived gland constituents like 3 or 4 and occupy defined positions in the tree constructed? Former studies Based on three genes (COI, 16S rRNA and Histone), our dealing with distribution of gland alkaloids in Stenus study provides further and comprehensive insights into the species revealed a correlation between gland composi- molecular phylogeny of the subfamily of Steninae, tradi- tions and different adaptions of beetles to their habitat tionally comprising the genera Stenus and Dianous. Results and threats like predation and infestation by microor- indicate that Dianous belongs to Stenus, confirming the ganisms (Lusebrink et al. 2008a, 2009;Langetal.2012; findings of Koerner et al. (2013) based on a single gene Schierling et al. 2013). Every alkaloid constituent pos- (COI). Monophyletic groups based on various morpho- sesses defined functions and exhibits certain logical characters introduced by Puthz (2001, 2008) are effectiveness to various factors. For instance, S. comma supported in some cases by topology of our constructed possessing chemotaxonomically basal alkaloids 1 and 2 trees. Furthermore, we show that chemotaxonomic (Schierling et al. 2013) lives directly adjacent to open approach presented by Schierling et al. (2013) is consistent waters (Horion 1963; Dettner 1987). Therefore, it is with molecular data obtained in this study. However, future almost certain that the beetle attains into water and could investigations using a wider range of Stenus and Dianous be threatened by drowning or potential predators like species will be necessary to fully reveal the molecular water striders (Linsenmair 1963). To avoid the danger, phylogeny of Steninae. glands of S. comma are equipped with spreading active gland compounds 1 and 2 (Schildknecht et al. 1975; Acknowledgments We gratefully acknowledge the Deutsche Forschungsgemeinschaft for funding this project (DE 258/12-1 and Lang et al. 2012), which enable the beetles to skim SE 595/14-1). Special thanks go to Dr. A. Kleeberg, Berlin for pro- rapidly over the water surface to escape these threats viding rare Stenus species for molecular analyses. Furthermore we (Piffard 1901; Billard and Bruyant 1905; Jenkins 1960; would like to thank Dr. S. Kehl, Department of Animal Ecology II, Schildknechtetal.1975). Epoxypiperideine group University of Bayreuth and S. Werner, Department of Mycology, University of Bayreuth for their precious help with software and tree member S. solutus is characterized by a cicindeloine- construction, respectively. Finally, thanks go to PD Dr. Joseph based secretion composition. In former studies, Woodring for language correction. 123 Phylogenetic relationships and chemical evolution 23

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