75 (2): 175 –183 8.9.2017 © Senckenberg Gesellschaft für Naturforschung, 2017. Cytogenetic comparison between Terrobittacus implicatus and Bittacus planus (Mecoptera: Bittacidae) with some phylogenetic implications Ying Miao & Bao-Zhen Hua * State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Plant Protection Resources and Pest Management of Education Ministry, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China; Bao-Zhen Hua * [[email protected]] — * Corresponding author Accepted 22.ii.2017. Published online at www.senckenberg.de/arthropod-systematics on 30.viii.2017. Editors in charge: Monika Eberhard & Klaus-Dieter Klass Abstract Cytogenetic data contribute greatly to taxonomic and phylogenetic analyses in many insect groups. However, such data have been largely neglected in Mecoptera to date. In the present investigation, we compared the meiotic progresses and karyotypes of the hangingflies Ter­ robittacus implicatus (Huang & Hua, 2006) and Bittacus planus Cheng, 1949 (Bittacidae) using C-banding technique and DAPI (4’-6-di- amino-2-phenylindole) and Giemsa staining. The karyotypical analyses show that T. implicatus possesses the highest chromosome number (2n = 41) ever observed in Bittacidae and an asymmetric karyotype with metacentric, submetacentric, subtelocentric and telocentric chro- mosomes. B. planus has a high diploid number (2n = 35), but a nearly symmetric karyotype with mainly metacentric and submetacentric chromosomes and a subtelocentric pair. Chiasmate meiosis and X0 sex determination mechanism are likely plesiomorphic in Bittacidae. The pronounced variations in cytogenetic traits within and between the genera Terrobittacus Tan & Hua, 2009 and Bittacus Latreille, 1805 suggest that they are substantial traits both for species delimitation and systematic analysis. The potential utilization of cytogenetic data for understanding the phylogeny of Bittacidae is briefly discussed. Key words Karyotype, chromosome number, meiosis, Terrobittacus, Bittacus, taxonomy, evolution. 1. Introduction The chromosomes of eukaryotic organisms can provide et al. 2008), Hymenoptera (LORITE & PALOMEQUE 2010), uniquely important information for taxonomic and phy- Lepidoptera (LUKHTANOV et al. 2011), and Diptera (VI- logenetic analyses due to their evolutionary conservation COSO & BACHTROG 2013). In Mecoptera, however, the (DYER 1979; DOBIGNY et al. 2004; GOKHMAN & KUZNET- cytogenetics was studied mainly before the 1970s, and SOVA 2006). Chromosomal analyses may help reveal many families including Bittacidae had been neglected the evolutionary relationships of species or higher taxa for decades (NAVILLE & BEAUMONT 1934; COOPER 1951, (WHITE 1974; FARIA & NAVARRO 2010), and play an im- 1974; ULLERICH 1961; BUSH 1967; ATCHLEY & JACKSON portant role in differentiating sibling species (BICKMORE 1970; XU et al. 2013). 2001; GOKHMAN & KUZNETSOVA 2006). Cytogenetic data The family Bittacidae is commonly known as hang- have been well documented in many holometabolous ingflies with a cosmopolitan distribution, and consists of insect groups, including Coleoptera (CABRAL-DE-MELLO 18 extant genera (CHEN et al. 2013). Bittacus Latreille, ISSN 1863-7221 (print) | eISSN 1864-8312 (online) 175 Miao & Hua: Cytogenetics of Bittacidae 1805 is the most speciose genus, and was regarded as 2.3. Cytogenetic analysis a repository for many “non-outstanding” species in Bittacidae (LAMBKIN 1988). Terrobittacus Tan & Hua, Chromosome spreads were prepared from the male tes- 2009 comprises four species endemic to China (TAN & tes of the fourth-instar larvae, pupae and newly-emerged HUA 2009). The phylogenetic position of Bittacidae in adults following IMAI et al. (1988). Testes were dissected Mecoptera is still a controversial problem. Bittacidae and submerged in fresh hypotonic KCl solution (0.045 was treated as a basal taxon based on morphological M) for 20 min at room temperature. After a short fixation characters (WILLMANN 1987), but was regarded as a sister of 30 – 40 s in acetic-ethanol (1 : 3, v/v), the testes were group to Panorpodidae based on molecular data (WHIT- transferred to a drop of 45% acetic acid on slides and torn ING 2002). into small pieces. The slides were air-dried for 24 h and The cytogenetic data of Bittacidae were considerably then treated with Sørensen’s phosphate buffer (pH 7.0) at scarce, with only three species of Bittacus having been 60°C for 30 min before staining (PIJNACKER & FERWERDA reported so far. MATTHEY (1950) was the first to discov- 1984). er that the males of B. italicus (Müller, 1766) had low C-banding was performed using the technique of chromosome number (2n = 25), chiasmate meiosis, and KING (1980). Air-dried slides were placed in HCl solu- an X0 sex determination mechanism. ATCHLEY & JACK- tion (0.2 M) for 30 min at room temperature, rinsed in SON (1970) provided the cytological observations of B. distilled water and dried. The slides were then placed in pilicornis Westwood, 1846 (2n = 29) and B. stigmaterus saturated Ba(OH)2 solution at 60°C for 3 min, dipped Say, 1823 (2n = 31), and proposed that the differences briefly in HCl and rinsed in distilled water. Afterwards, in chromosome number were crucial characters for dif- the slides were placed in Sørensen’s phosphate buffer ferentiating Bittacidae from Panorpidae. (pH 7.0) at 65°C for 30 min, rinsed in distilled water and In this paper, we present information on karyotypic stained in 5% Giemsa for 15 min. Slides were then rinsed details and male meiosis of the hangingflies Terrobitta­ in distilled water and dried. Some air-dried slides were cus implicatus (Huang & Hua, 2006) and Bittacus planus subjected to fluorescent staining with DAPI (4’-6-di- Cheng, 1949 for the first time, in an attempt to contribute amino-2-phenylindole) for 3−5 min at room temperature the cytogenetic data of Bittacidae for future systematic (REBAGLIATI et al. 2003). Photographs were taken with a analysis. Nikon DS-Fil digital camera mounted on a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). The fluorescent signals were observed with UA filter (330 – 385 nm) for the fluorochrome DAPI. 2. Materials and methods 2.4. Statistical analyses 2.1. Biological materials At least five spermatogonial cells with well-spread chro- Adults of Terrobittacus implicatus and Bittacus planus mosomes at metaphase were used to measure the sta- were collected from the Huoditang Forest Farm (33°26′N tistics of chromosomes for each species. The captured 108°26′E, elev. 1570 – 1600 m) in the Qinling Moun- images were quantified using the NIS-Element D 3.22 tains, Shaanxi Province, China from late July to August software (Nikon, Tokyo, Japan). For each chromosome, in 2015. centrometric index (i = the length of short arm/the length of chromosome), arm ratio (r = the length of long arm × 100 / the length of short arm), and means and standard 2.2. Insect Rearing deviations (SD) of absolute length (AL = actual length of chromosomes) and relative length (RL = absolute Live adults were reared in screen-wired cages (40 cm × length of chromosome × 100 / total length of the haploid 60 cm × 60 cm) furnished with fresh plant twigs for rest- complement) were calculated using a Microsoft Excel ing and moist absorbent cotton for drinking and keeping spreadsheet (Table 1). The centromeric nomenclature humidity (GAO & HUA 2013; JIANG et al. 2015). Eggs, system adopted LEVAN et al. (1964). Stebbins’ type and larvae, and pupae were incubated or reared in plastic con- asymmetry index methods were used to assess the degree tainers with humid humus. Live Musca domestica adults of karyotype asymmetry (Table 2). Stebbins’ type be- were provided as food items for the adults, and freshly- longed to a qualitative category, and was established by killed Tenebrio molitor larvae were given to the larvae. recognizing three degrees of difference (A – C) between The eggs, larvae and pupae were reared in the laboratory the largest and smallest chromosome of the complement, from October 2015 to May 2016. The temperature was and four degrees of proportion (1 – 4) of chromosomes kept at 16 ± 2°C for the larvae, 21 ± 2°C for the pupae that are metacentric with an arm ratio of less than 2 : 1 and 23 ± 2°C for the adults. The relative humidity was (STEBBINS 1971). Asymmetry index (AI) was a quantita- 75 ± 10%. tive parameter, refering to the coefficient of variation of centromeric index (CVCI) × coefficient of variation of chromosome length (CVCL)/100 (PASZKO 2006). 176 ARTHROPOD SYSTEMATICS & PHYLOGENY — 75 (2) 2017 3. Results chiasma. In T. implicatus chiasmata range from 11 to 20 in 50 nuclei counted, with a mean chiasma frequency of 15.3 per nucleus and a mean chiasma frequency of 0.8 3.1. Karyology per autosomal bivalent. None of the nuclei observed ex- hibits ring-shaped bivalents. Terrobittacus implicatus (Fig. 1A) and Bittacus planus Bivalents assemble at the equatorial plate and become (Fig. 2A) exhibit different chromosome numbers and oriented with their centromeres poleward at metaphase I karyotype morphologies (Figs. 1B,C, 2B,C; Tables 1, 2). (Fig. 4A). The two centromeres are oriented in the long T. implicatus possesses 2n = 41, with a karyotypic axis of the spindle equidistant from the equator, and the formula of 5m + 6sm + 2st + 28T, and a fundamental chiasmata are located in the equatorial plate. Three kinds number of total chromosome arms (FN) of 54 (Fig. 1C, of bivalents are frequently observed at the metaphase Table 1). T. implicatus has an asymmetric karyotype with and anaphase I: M-shaped or half-ring (metacentric bi- AI of 32.93 (Table 2). The proportion of chromosomes valent with one terminal chiasma, or with two chiasmata with arm ratio (r) less than 2 : 1 is 0.14 (Table 1), belong- but one releasing first; arrowheads in Fig. 4A,B), cross- ing to the third degree (1.50 – 0.01). The ratio between shaped (autosomal bivalent with one interstitial chiasma; the largest and smallest chromosome of the complement open arrows in Fig. 5A,B), and rod-shaped (autosomal is 2.09, corresponding to type B (2 : 1 – 4 : 1).