Diptera) and the Cost of Male Copulations in Saltella Sphondylii

Org Divers Evol (2011) 11:253–261 DOI 10.1007/s13127-011-0054-2

ORIGINAL ARTICLE

New information on the evolution of mating behaviour in Sepsidae (Diptera) and the cost of male copulations in Saltella sphondylii

Denise Siew Hoong Tan & Sheng Rong Ng & Rudolf Meier

Received: 24 December 2010 /Accepted: 26 July 2011 /Published online: 13 August 2011 # Gesellschaft für Biologische Systematik 2011

Abstract Here we describe the hitherto unknown Introduction details of the highly unusual mating behaviour of Saltella sphondylii—a widely cited model for male TheSepsidae(“black scavenger flies”)are,withap- longevity costs caused by multiple copulations. When proximately 320 described species, a moderately large compared to the known mating behaviour of 28 sepsid family of acalyptrate flies (Diptera: Cyclorrhapha: species, we find five unique behavioural elements based Schizophora) occurring in all zoogeographic regions on frame-by-frame analyses of video-recordings. These (Ozerov 2005). Many species are attracted in large new behaviours are documented with video clips. We numbers to dung, carrion, and other decaying organic suggest that the male longevity costs could be due to substrates where they can be recognized readily based on copulation bouts that involve multiple insertions of a an ant-like habitus that is caused by the constriction of the comparatively membranous phallus into the female. We first two abdominal segments (Pont and Meier 2002). At compare the phallus of the Saltella sphondylii to those the substrate, the females will feed and oviposit while the from three other species (Themira putris, Parapaleosepsis males attempt to copulate with females. Many sepsid plebeia, Sepsis punctum). species are common and breed under laboratory con- ditions, which have made them attractive study objects for Keywords Sepsidae . Mating cost . Mating behaviour . biologists working in different fields ranging from the Longevity. Polyandry. Sexual conflict evolution of sexual dimorphisms (Ang et al. 2008; Blanckenhorn et al. 2007;Eberhard2001a, b, 2002a, b; Ingram et al. 2008; Puniamoorthy et al. 2008), over behaviour (Parker 1972a, b), systematic theory (Laamanen et al. 2005;Meier1997) to developmental biology (Bowsher and Nijhout 2007, 2009), and genomics (Hare Electronic supplementary material The online version of this article et al. 2008). (doi:10.1007/s13127-011-0054-2) contains supplementary material, Many species are strongly sexually dimorphic with which is available to authorized users. regard to forelegs and sternites. The forelegs of males are ’ D. S. H. Tan : S. R. Ng : R. Meier modified for grasping the females wings before and/or Department of Biological Sciences, during copulation (Eberhard 2001b; Ingram et al. 2008; National University of Singapore, Puniamoorthy et al. 2008) while the 4th sternites are often 14 Science Drive 4, modified into brushes that are used to stimulate females. Singapore 117543, Singapore The study of sepsid behaviour started with Hammer (1941) R. Meier (*) who described an “elegant minuet-like” dance in Sepsis University Scholars Programme, duplicata, and Parker (1972a, b) who described an unusual National University of Singapore, precopulatory guarding behaviour for Sepsis cynipsea. This 14 Science Drive 4, Singapore 117543, Singapore species subsequently became widely studied (Blanckenhorn e-mail: [email protected] et al. 2000, 2002; Martin and Hosken 2002; Muhlhauser 254 D.S.H. Tan et al. and Blanckenhorn 2002, 2004; Ward et al. 1992) but it was Materials and methods not until 20 years later that the behaviour of several additional species was described (Eberhard 1999, 2001a, Virgin S. sphondylii flies were separated by sex within 24 h 2002a, c, 2003, 2005; Eberhard and Pereira 1996; of eclosion from a laboratory culture established using Puniamoorthy et al. 2009). This comparative research females collected in Kevelaer (Germany) and maintained as was supported by a better understanding of the phyloge- described in Puniamoorthy et al. (2009). In order to obtain netic relationships within the family (Meier 1995a, b, sexually mature flies, the sexes were housed separately for 1996;Suetal.2008). a period of 5 days before being used for mating trials. Due For several decades, behavioural research on Sepsidae to high mortality in the parental culture, only a relatively was based largely on macroscopic observations although small number of virgins were available for experiments (n= most species are too small (<5 mm) and move too fast 14 individuals) that involved one male and one female. to observe the details without the use of a microscope. Note that, based on previous experience (see Puniamoorthy Recently, observing and/or video-taping mating behav- et al. 2009), a small number of successful mating trials is iour through microscopes has revealed new insights sufficient for describing the qualitative elements of sepsid (Ang et al. 2008;Eberhard2001a, b, 2002a, b, c, 2003), mating behaviour. Recordings began upon the introduction and demonstrated that the behaviour of sepsids is of both flies into the same Petri dish and were terminated surprisingly diverse (Puniamoorthy et al. 2009). Many after the male voluntarily dismounted from the female and insects rely heavily on olfactory and auditory species- the pair remained separate for more than 1 min. The specific cues (Doi et al. 2001;Freyetal.1992; Henry et recordings were analysed frame-by-frame (1 frame=1/25th al. 2002), but Puniamoorthy et al. found that in sepsids of a second) and behavioural elements were noted, even the visible male courtship behaviour was species- categorized and described. We coded known behaviours as specific. Saltella sphondylii (Schrank 1803)wasnot in Puniamoorthy et al. (2009), while new behaviours were included in Puniamoorthy et al.’s studies although Martin either coded as new states or new characters. We then and Hosken’s(2004) and Schulze’s(1999)macroscopic mapped the 33 characters (Table 1, and see character observations suggested that the mating behaviour of this descriptions in Appendix) on the molecular phylogenetic species might be very unusual. They noticed postcopula- hypothesis in Puniamoorthy et al. (2008) with the exception tory guarding, a lack of female resistance to mating, no of Toxopoda sp., which was missing in this analysis and overt courtship behaviour, and short and intermittent whose position was based on Su et al. (2008). Character copulations (Martin and Hosken 2004:358:“i.e., males optimisation procedures follow Puniamoorthy et al. (2009). engage and disengage genitalia”). This contrasts sharply In order to determine levels of homoplasy, we recorded tree with other sepsid species that lack postcopulatory guarding, length and consistency index with and without S. sphondylii. have strong female resistance, extensive courtship In addition to the behavioural research, we also extracted behaviour, and engage genitalia only once in a copulation the phallus of male S. sphondylii, Themira putris (Lin- bout (Puniamoorthy et al. 2009). Here, we describe the naeus, 1758), Parapaleosepsis plebeia (de Meijere, 1906), behaviour in detail and update a matrix of behavioural and Sepsis punctum (Fabricius, 1794) and studied their characters published in Puniamoorthy et al. (2009) morphology under a scanning electron microscope (SEM). Additional information on S. sphondylii is particularly These species represent major branches on the sepsid tree important because life history costs related to courtship (Fig. 1) and copulation have been extensively documented and quantified mostly for females while fewer authors have investigated male costs (but see Burton-Chellew et al. Results and discussion 2007; Cordts and Partridge 1996; Ferkau and Fischer 2006; Kotiaho and Simmons 2003; Oliver and Cordero The mating behaviour of S. sphondylii is very unusual for 2009; Paukku and Kotiaho 2005; Prowse and Partridge sepsids in that males in all mating trials copulated 2–3 1997; South et al. 2009). One prominent exception is times over a period of 10–18 minutes without dismounting Martin and Hosken’s(2004) demonstration of a negative the female. The copulation duration is the shortest (3– correlation between the number of copulations and 4 mins) known for any sepsid species. Note that we only longevity in S. sphondylii males. On average, virgin males count periods of genital contact that lasted for more than lived 28 days while a single copulation reduced longevity 2 mins as proper copulations. In between these copulations by4days,twoby6,fourby8,andsixby12days. up to five more sporadic and short instances of genital However, Martin and Hosken’s(2004) description lacks contact occurred that lasted from a few seconds to just over information on behavioural details that can be observed 1 min. At least some of these interactions included the only under a microscope. insertion of male genitalia into the female. Martin and oto aecpltosin copulations male of Cost

Table 1 Behavioural character matrix modified from Puniamoorthy et al. (2009) with new information for Saltella sphondylii

List of Species Behavioural characters

123456789101112131415161718192021222324252627282930313233 atlasphondylii Saltella Allosepsis indica 0000221220 –– –000 00000 0– 00– 00– 00 00 Australosepsis frontalis 10001– 10–– – – – 000 10000 0– 02– 00– 00 00 Australosepsis niveipennis 10001– 0101 02 0000 00000 11 02– 0100000 Dechaetaphora aeneipes 0000201102 – 2 1200 ?0003 10&102– 0100100 Dicranosepsis sp.1 10001– 1 1 0 0&1 0 0 0 0 0 1&3 0 0 0 0 0 1 1 1 0 – 0100000 Dicranosepsis sp.2 10001– 1 1 0 0&1 0 2 0 0 0 1&2 0 0 0 0 2&3 1 1 1 0 – 0100000 Meroplius fukuharai 0000211100 02 0012 00000 11 000010 00 00 Nemopoda nitidula 00001– 1201 –– –000 00000 0– 000010 02 00 Orygma luctuosum 00100– 0220 –– –002 00100 0– 00– 1102000 Parapaleosepsis plebeia 00001– 1111 12 1001 20004 12 00– 0100020 Perochaeta dikowi 01000– 10–– – – – 110 20000 10&3– 3110– 00 00 Sepsis cynipsea 1000201101 02 0000 20003 10&102– 0100310 Sepsis dissimilis 10001– 0111 12 0000 00010 10&102– 0100000 Sepsis flavimana 0002211110 – 2 0002 00001 11 00– 1100000 Sepsis pyrrhosoma 0001211110 – 2 0202 00001&311 02– 0100000 Sepsis neocynipsea 0000200101 02 0101 00004 11 00– 0 1 0 0 0&3 1 0 Sepsis nitens 00001– 1110 – 2 0000 00001 11 00– 0100000 Sepsis punctum 10022110–– – – – 000 00000 10&120– 0100000 Sepsis secunda 11000– 00–– – – – 000 00000 10 – 0 – 0100100 Themira annulipes 0001???101 11 1000 00133 0– 020?11 00 00 Themira biloba 0010210111 02 1112 00101&310 020010&101 00 Themira flavicoxa 0000210111 02 0100 11100 10&2000010&101 00 Themira lucida 0000210111 02 1102 20102 10 010110 01 00 Themira minor 0000200101 00 1111 20100 10 010010 01&200 Themira putris 0012210121 12 1103 00101 10 021110 00 00 Themira superba 0000210101 10&21102 00002 10 001010 00 00 Toxopoda sp. 00001– 00–– – – – 010 00020 14 00– 0 1 0 1 2&3 0 0 Saltella sphondylii 00021– 200––––100 – 0201 12&301– 2100100&1 255 256 D.S.H. Tan et al.

Willistoniella pleuropunctata 3 8 9 16 27 30 Orygma luctuosum 0>1 1>2 0>2 0>2 0>1 0>2 8 19 31 Nemopoda nitidula 1>2 1>0 1>2 4 11 20 21 25 29 31 13 Themira annulipes 0>1 0>1 0>1 0>3 1>3 1>2 0>1 1>0 6 12 15 17 21 5 7 21 25 Themira minor 1>0 1>0 0>1 0>2 1>0 1>2 1>0 0>1 0>1 15 14 16 22 3 25 Themira biloba 0>1 0>1 0>1 0>1 4 9 11 16 26 27 31 0>1 1>2 Themira putris 9 12 16 0>2 1>2 0>1 2>3 0>1 0>1 1>0 9 11 19 26 31 5 7 10 31 0>1 1>2 1>2 Themira superba 21 25 1>0 0>1 1>0 0>1 1>0 0>1 0>1 0>1 0>1 13 16 18 21 1>2 1>0 17 Themira flavicoxa 1>0 2>0 0>1 2>0 17 25 27 4 7 14 19 21 23 25 27 0>1 Themira lucida Saltella sphondylii 1>2 0>1 0>1 0>2 1>2 0>1 1>2 0>1 1>2/30>1 0>2 6 10 13 14 15 21 25 5 8 Dechaetaphora aeneipes 1>0 1>2 0>1 0>2 1>0 0>3 0>2 10 16 31 8 12 22 23 1>2 0>1 Meroplius fukuharai 1>0 0>2 1>0 1>0 1>2 0>1 0>1 7 20 23 30 31 15 19 Toxopoda sp. 1>0 0>2 1>4 0>1 1>2/3 0>1 1>0 2 5 14 17 25 27 28 Perochaeta dikowi 0>1 1>0 0>1 0>2 0>3 0>1 1>0 26 5 8 9 10 16 22 28 1 9 11 13 23 Allosepsis indica 0>1 1>2 1>2 1>2 1>0 1>0 1>0 1>0 17 21 32 1>0 0>1 0>1 0>1 1>2 6 16 Parapaleosepsis plebeia 31 0>2 0>4 0>2 12 1>2 0>1 1>0 24 Dicranosepsis sp. 1 2>0 21 0>1 Dicranosepsis sp. 2 0>2/3 7 20 25 9 11 Sepsis dissimilis 1 8 15 1>0 0>1 1>2 1 10 21 25 0>1 0>1 0>1 0>1 1>0 25 Sepsis nitens 1>0 1>0 0>1 1>0 8 17 22 28 0>1 25 Australosepsis frontalis 1>0 0>1 1>0 1>0 7 1>2 Australosepsis niveipennis 1>0 2 5 7 8 23 31 Sepsis secunda 9 10 0>1 2>0 1>0 1>0 1>0 0>1 4 27 0>1 1>0 1 4 16 21 Sepsis flavimana 1>2 0>1 14 25 5 1>0 0>1 0>2 0>1 Sepsis pyrrhosoma 0>2 0>2 1>2 4 8 24 Sepsis punctum 0>2 1>0 0>2 17 25 31 6 21 31 32 Sepsis cynipsea 0>2 0>2 1>3 1 7 14 16 21 1>0 0>3 0>1 0>1 Sepsis neocynipsea 1>0 1>0 0>1 0>1 3>4

Fig. 1 Evolution of behavioural characters modified from Puniamoorthy et al. (2009) on the phylogenetic tree of Sepsidae based on the molecular data from Puniamoorthy et al. (2008) and Su et al. (2008) (ACCTRAN: black symbols non-homoplasious; open symbolshomoplasious changes)

Hosken (2004) counted the period from successful male that it has a more membranous and intricate terminal mounting to the natural dismount as one copulation. Based endophallus than other sepsid species (see arrows on on our observations each copulation sensu Martin & Fig. 2). The morphology of the male phallus suggests that Hosken is thus likely to have involved 3–5 insertions and a more rigorous test of this damage hypothesis based on retractions of the phallus into and from the female. Inserting experiments with single- and multiple-mated males would the phallus multiple times is very atypical for sepsids that be promising. Such experimental work may clarify the normally require only one insertion per successful copu- proximate causes for the high male mortality, but it would lation. It is thus conceivable that the number of genital raise additional, ultimate questions with regard to why a contacts of Saltella males in a single copulation bout (10– membranous phallus evolved and why it is inserted 18 mins) is similar to what most sepsid males will multiple times during a copulation bout. encounter during their lifetime. This raises the question whether the large number of insertions could be the Courtship behaviour proximate cause for the high male mortality (e.g. through damage to the male phallus). A first assessment of this Frame-by-frame analyses of high magnification recordings hypothesis can be based on the study of the male and revealed that the courtship behaviour repertoire of S. female reproductive organs. Puniamoorthy et al. (2010) sphondylii is extensive, with different displays being used described the female reproductive organs across Sepsidae before, during and in between proper copulations (Table 2, and found no obvious structures in the female reproductive see video clips in supplementary information and on tract of Saltella that could explain the higher mortality of Youtube). Unless mentioned otherwise, all of the following males. However, the phallus of S. sphondylii is atypical in precopulatory and copulatory behaviours were constant Cost of male copulations in Saltella sphondylii 257

Fig. 2a–d Scanning electron microscopy (SEM) phallus images. a Saltella sphondylii; b Themira putris; c Parapaleosepsis plebeia; d Sepsis punctum (scale=0.1 mm). Arrows indicate the membranous phallus elements and thin spines

throughout all mating pairs observed. We find five unique rubbing female’s head and female’s abdomen (see video features (coded as four new character states and one new links in Table 2). Behaviours performed in between copu- character) compared to the 33 behavioural characters in the lation are more variable with regard to the sequence of study by Puniamoorthy et al. (2009). For example, when events and their duration. For example, the violent head the male is mounted on females, his mid legs are neither in thumping behaviour was only performed by 4 of the 7 males. contact with the female nor stretched out away from the We can also confirm Puniamoorthy et al.’s(2009)finding female’s body (described in character 7 in Puniamoorthy et that mating behaviour is highly homoplastic. The addition of al. 2009). In all trials, the males’ mid- and hind legs walked Saltella to the matrix yielded one new behavioural character along with the female (no direct contact) instead of being and four new states. When the data were mapped onto the rested on the females’ wings, dragged along the substrate or molecular tree, we found that, compared to Puniamoorthy et lifted off the ground (described in characters 17 and 19). al. (2009), the tree length increased by ten steps and the Mated couples always separated after copulation without a consistency index (CI) by 0.02 (from 0.28 to 0.30; Fig. 1). 180° turn, allowing the male to remain mounted in order to The low CI value indicates that homoplasy is rampant and re-initiate courting and re-establish genital contact (de- higher than for the third positions for the protein-encoding scribed in character 27). The intensity at which males tap genes used for reconstructing the tree (CI=0.33, see the female’s head with both mid legs is far greater than what Puniamoorthy et al. 2009). had been previously observed in Dechaetaphora aeneipes Overall, our study of Saltella sphondylii illustrates that it (de Meijere, 1913) and Sepsis pyrrhosoma Melander and is important to use microscopic techniques when studying Spuler, 1917 (new character 33; compare video for the mating behaviour of sepsids. Details are revealed that courtship behaviour between copulations with video evi- are easily overlooked in studies using macroscopic techni- dence in Tan et al. 2010). Several behaviours are shared ques. Microscopic techniques are also needed in order to between S. sphondylii and other sepsids (see Puniamoorthy reveal the bewildering diversity of sepsid mating behaviour. et al. 2009). These include surstylus stimulation, tapping of Once a male is anchored on a female with its forelegs, it the ventral side of the female’s abdomen by male hind legs, can only use the mid- and hind legs, abdomen, and head for male proboscis interaction with the dorsal region of the mating behaviours. Yet, it appears that the males of all female’s thorax, rubbing of female’s head using one or both sepsid species have found unique ways to use these body mid-tarsi, male mid legs rubbing male hind legs first then parts to entice females into copulation. 258 D.S.H. Tan et al.

Acknowledgements We would like to thank all members of the Evolutionary Biology Lab, especially Martin Chew for his assistance in generating the molecular phylogeny, and Nalini Puniamoorthy for her helpful comments for the manuscript. This study was financially supported by grant R154-000-476-112 from the Ministry of Education in Singapore.

Appendix http://youtu.be/s3bd4uvsmxk http://youtu.be/vSC15ZzQ1R8 http://youtu.be/KfpqxifmhUY

Character descriptions modified from Puniamoorthy et al. (2009)

(1) ‘Circling’—0: Absent (Male approach without gliding motion); 1: Present (Male circling female in a gliding motion, head and thorax leading the change of direction with abdomen bent at an angle) genital contact is broken (2) — Characteristics Video Links Initial mount 0: Male jumps or climbs onto female; 1: Male bends abdomen anteriad to establish direct genital contact (3) Effect of struggle on in-copula position of pair—0: Females do not flip over; 1: Pair flipping over resulting in male on his back and female with her legs in air) (4) Male proboscis–female interaction—0: No contact between male proboscis and female; 1: Male extends proboscis to ‘kiss’ female ocelli; 2: Male extends proboscis to tap dorsal part of female thorax (5) Male grasp of female wingbase—0: Forelegs resting on female thorax; 1: Forelegs release wingbase only at separation; 2: Forelegs release female wings well s head (character 22 and 23; state 1, 2)s abdomen (character 22 & 23; Occurs state just 1, before 3) Prolonged

’ ’ before separation (6) Male foreleg after release—0: Resting against female

s head (character 33; state 0&1) Occurs forcefully thorax (no movement); 1: Male foreleg interacting ’ with female thorax and, or foreleg (7) Midleg position—0: Male midlegs always in contact with female; 1: Male mid legs are stretched out away from female body i.e. ‘balancing’ (for extended

males before, during and between copulations with links to corresponding videos period); 2: Male midlegs are not stretched out and are not in contact with female (8) Motion restricted to mid tarsus—0: No independent tarsal movements; 1: Curling (movement of the tarsi S. sphondylli 2–4 against the barsitarsus); 2: Quiver (vibration of entire tarsus without movement of tibia or femur) (9) Non-contact midleg movement—0: Simultaneous usage of both midlegs; 1: Simultaneous and alternate; 2: Alternate usage of both midlegs (10) — Surstylus stimulation (character 25; state 1)Male uses one or bothMale mid-tarsi rubs to hindlegs rub with female its head midlegs (character and 14; then state rubs 1) female Male rubs hindlegs with its midlegs and then rubs female Prolonged and persistent Brief Male uses one or bothMale mid-tarsi raises to both rub midlegs female high head and (character thumps 14; female state 1)Direction Prolonged of midleg movement 0: Male midleg stretched out and stationary; 1: Male midleg towards female eye; 2: Male midlegs move posteriad (11) Male midleg movement away from female head—0: Smooth return without any interruptions; 1: Return Behavioural elements performed by interrupted by midleg waves (12) Midleg rotation during tarsal curl—0: Midleg curling in a horizontal plane; 1: Curling direction shifting Table 2 Behavioural elements Copulatory Male proboscis taps dorsal part of female thorax (character 4, state 2) Prolonged Pre-copulatory Male hindlegs tap ventral side of female abdomen (character 21; stateBetween 1) Copulations Surstylus stimulation (character 25; state 1) Brief Prolonged Cost of male copulations in Saltella sphondylii 259

from horizontal to vertical plane through leg rotation; stimulate female close to her genital opening; 3: Male 2: Curling in a vertical plane lowers surstylus to stimulate on ventral surface of (13) Number of tarsal curls per midleg movement—0: female abdomen Single curl per midleg movement; 1: Multiple curls (26) Male tapping female with modified fourth sternites— (14) Midleg interaction with female head—0: No with 0: Modified 4th sternites of males used to tap or contact female head; 1: Male uses his mid tarsi to rub stroke dorsal part of female abdomen; 1: Ventral part head; 2: Male uses his mid tarsi to tap head (singular of female abdomen movements) (27) Separation after copulation—0: Quick (one or two (15) Midleg interaction with female abdomen—0: No 180° turns by the male); 1: Long (involving a with contact female abdomen; 1: Male uses his prolonged struggle between male and female in midlegs to tap female abdomen trying to break genital contact); 2: Quick but not (16) Midleg interaction with female thorax—0: Absent; 1: involving turns by the male Midlegs tap lateral surface (singular movements); 2: (28) Female Shake—0: Absent (no violent side to side Midlegs ‘stroke’ lateral surface (extended rubbing); movement); 1: Present 3: Midlegs ‘stroke’ dorsal surface (extended rubbing) (29) Type of female shake—0: Horizontally; 1: Vertically (17) Female wings bent down by male midlegs—0: Male (30) Female foreleg movements—0: No significant move- midlegs rest on female wings; 1: Male midlegs used ments of forelegs; 1: Female intermittently lifts to forcibly bend down female wings forelegs off the substrate; 2: Female repeatedly lifts (18) Midleg to midleg grasp—0: Male midlegs not forelegs above head holding female mid legs; 1: Male uses midlegs to (31) Female hindleg movements—0: Female hindlegs not hold onto female midlegs used to interact with male; 1: Female hindleg used to (19) Contact of male hindleg with substrate—0: Hindleg ‘kick’ male; 2: Female hindleg ‘rubbing’ male hindlegs not in contact with substrate when female moves; 1: (32) Female ejection of ovipositor when male is Mounted male’s hindleg is dragged along substrate mounted—0: Absent; 1: Female ejects ovipositor when when female moves; 2: Mounted male walks in after genital contact; 2: Female ejects ovipositor prior to tandem with female instead of being dragged genital contact (20) Non-contact movement of male hindlegs—0: Absent; (33) Use of male midleg—0: No forceful contact with 1: Hindlegs curl backwards 360° like a backward female head; 1: Male uses midleg to ‘beat’ female head ‘butterfly stroke’;2:‘Cycling’ ( i.e. like a peddling motion); 3: Curling (Hindlegs stretched out and vibration of the 2–4 tarsal segments against the References barsitarsus) (21) Usage of hindlegs—0: No direct contact with female; 1: Tap ventral side of female abdomen; 2: Rub Ang, Y., Puniamoorthy, N., & Meier, R. 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