Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(4): 111-120, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-966 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: M. Husemann, D. Sadílek, L.-S. Dey, O. Hawlitschek, M. Seidel (2020) New genome size estimates for band- winged and slant-faced grasshoppers (Orthoptera: Acrididae: Oedipodinae, Gomphocerinae) reveal the so far larg- est measured insect genome. Caryolo- gia 73(4): 111-120. doi: 10.13128/caryolo- gia-966 Received: June 10, 2020 Accepted: September 24, 2020 Published: May 19, 2021 Copyright: © 2020 M. Husemann, D. Sadílek, L.-S. Dey, O. Hawlitschek, M. Seidel. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. New genome size estimates for band-winged and slant-faced grasshoppers (Orthoptera: Acrididae: Oedipodinae, Gomphocerinae) reveal the so far largest measured insect genome Martin Husemann1,*+, David Sadílek2+, Lara-Sophie Dey1, Oliver Hawlitschek1, Matthias Seidel1,3 1 Centrum für Naturkunde, Universität Hamburg, Martin-Luther-King-Platz 3, DE-20146 Hamburg, Germany 2 Department of Zoology, Faculty of Science, Charles University, Viničná 7, CZ-12843 Praha, Czech Republic 3 Department of Entomology, National Museum in Prague, Cirkusová 1740, CZ-19300 Praha, Czech Republic *Corresponding author. E-mail: martin.husemann@uni-hamburg.de +MH and DS equally contributed Abstract. Grasshoppers, specifically those of the family Acrididae are known to have the largest genomes of all insects. However, less than 100 species of Orthoptera have their genome size estimated so far. In the present study, we measured the genome size of five acridid species belonging to the two subfamilies Oedipodinae and Gomphoceri- nae. All of the genomes measured are large and range between 1C = 11.31 pg in the female of Chorthippus dorsatus and 1C = 18.48 pg in the female of Stethophyma gros- sum. The latter represents the so far largest measured insect genome. We further pro- vide a summary of genome size estimates available for Orthoptera. Keywords: C-value, flow cytometry, Stethopyhma, Oedipoda, Sphingonotus, Chorthip- pus. INTRODUCTION The genome has become one of the most important targets of interest for biologists. In times of high throughput sequencing, projects like i5k generate data of entire genomes are at a daily base (Robinson et al. 2011; Li et al. 2019). However, we still have little data and a limited understand- ing of the variance in genome size across organisms. Especially for insects, the most diverse group of organisms on earth, data of only about 1,300 of the expected diversity of several million species are available (Sadílek et al. 2019a; Gregory 2020). Generating new data on genome sizes is important, e.g., for choosing the adequate NGS applications for genomic sequencing (Rodríguez et al. 2017). Yet, genome size can also be a taxonomic feature 112 Martin Husemann et al. and can be used for species determination (Sadílek et al. 2019b). For many applications taxa with specifically large genomes still remain a difficult target, especially if no complete genome sequence is available. Further, in order to understand why some species or species groups have specifically large genomes, whereas others are rather small requires comprehensive data across a large range of taxa. While the so far largest genome of any organism was estimated in a plant, the monocot Paris japonica Franchet with 1C = 152.23 pg (Pellicer et al. 2010), the largest genome sizes in insects have been measured in Orthoptera, specifically Caelifera, with 1C values of 16.93 pg in Podisma pedestris (Linnaeus, 1758) (Podis- minae) and 16.34 pg in Stauroderus scalaris (Fischer von Waldheim, 1846) (Gomphocerinae) (Gregory 2020 for a list). However, there is also a lot of variation with- in Orthoptera with genome sizes as small as 1C = 1.55 pg found in the cricket Hadenoecus subterraneus (Scud- der, 1861) (Rasch and Rasch 1981). Nevertheless, a clear trend for larger genomes in the short-horned grasshop- pers is observed, and specifically in the family Acridi- dae. In the present study, we were able to locate only 85 published genome size estimates from all Orthoptera (e.g. Gregory 2020). To better understand the evolution of genome size in Orthoptera, especially the huge genomes of grasshop- pers of the Acrididae family, it is obligatory to generate additional information. Hence, we provide new genome size information for members of the Acrididae fam- ily, i.e. three species of the subfamily Oedipodinae and two species of the Gomphocerinae. We present, to our knowledge, the so far largest genome size of any insect and summarize the knowledge on genome sizes in Orthoptera. MATERIAL AND METHODS Sampling Eight specimens from five species (Table 1), all of the family Acrididae, were collected for our analyses in September 2019 in Hamburg, Georgswerder (Ger- many, 53.5097°N 10.0301°E). Specimens were collected by hand and kept alive until further processing. We included two species of the subfamily Gomphocerinae: Chorthippus dorsatus (Zetterstedt, 1821) and a species of the Chorthippus biguttulus (Linnaeus, 1758) group (a group of three species C. biguttulus, C. brunneus (Thun- berg, 1815), C. mollis (Charpentier, 1825), which can only be identified with certainty by male song patterns; our specimen is a female, but according to morphologi- cal traits most likely represents C. biguttulus), as well as three species of the subfamily Oedipodinae: Oedipoda caerulescens (Linnaeus, 1758), Sphingonotus caerulans (Linnaeus, 1767), and Stethophyma grossum (Linnaeus, 1758) (Table 1, 2). Reference specimens are deposited in the Zoological Museum Hamburg (ZMH), part of the Center of Natural History (CeNak) under the accession ZMH 2019/21. Genome size analysis Nuclear DNA content (2C) was measured by the f low cytometry method (FCM) as in Sadílek et al. (2019a, b) at the Department of botany of Charles Uni- versity, Prague. The muscle tissue of one hind femur was used for FCM analysis against the plant-internal stand- ard Pisum sativum L. “Ctirad” (Fabaceae) with 2C = 9.09 pg (Doležel et al. 1998; Doležel and Greilhuber 2010). Fresh tissue was homogenized and mixed with a leaf of Table 1. Diploid chromosome number, 2C genome size, sample/standard ratio of both DAPI- and PI-stained samples and GC content of grasshopper species studied. Samples were measured against P. sativum standard with 2C = 9.09 pg. F = female, M = male, 2n = male dip- loid chromosome number, 2C = nuclear DNA content for nuclei with diploid chromosome number, CV = average coefficient of variation for each stain used. Species 2n Sex 2C (pg) Sample/ standard DAPI ratio Sample/ standard PI ratio GC content (%) Sample CV DAPI - PI Sphingonotus caerulans 22+XX F 26.63 2.424 2.930 42.14 2.70 - 2.95 Sphingonotus caerulans 22+X0 M 25.12 2.321 2.764 41.87 2.71 - 2.81 Oedipoda caerulescens 22+XX F 28.39 2.621 3.123 41.88 3.71 - 5.62 Chorthippus dorsatus 16+XX F 24.14 2.359 2.656 40.82 2.58 - 2.64 Chorthippus biguttulus 16+XX F 22.62 2.149 2.488 41.35 2.50 - 4.07 Stethophyma grossum 22+XX F 36.95 3.326 4.065 42.35 3.41 - 4.23 Stethophyma grossum 22+X0 M 34.72 3.172 3.820 42.08 2.19 - 2.84 113New genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome Ta bl e 2. G en om e si ze s of O rt ho pt er a so fa r m ea su re d. Th e te m pl at e of t he t ab le w as e xt ra ct ed fr om G re go ry ( 20 20 ); it w as c om pl em en te d w ith o ri gi na l r ef er en ce s an d ad di tio na l s tu d- ie s. R ef er en ce s w ith a n * in di ca te t ha t th e or ig in al r ef er en ce c ou ld n ot b e ac ce ss ed a nd d at a ar e ex tr ac te d on ly f ro m G re go ry ( 20 20 ). 1 r el at iv e ge no m e si ze - m ea su re d w ith t he D A PI - fr om M or ga n- R ic ha rd s (2 00 5) . M = m al e, F = fe m al e, 2 C = g en om e si ze o f t he d ip lo id c el l, 2n = d ip lo id c hr om os om e nu m be r (i f s ex is n ot d et er m in ed , k ar yo ty pe o f t he m al e is p re - se nt ed ; i n al l s pe ci es t he s ex d et er m in in g sy st em is X X /X 0, o nl y m al es o f P od is m a pe de st ri s ca n be v ar ia bl e w ith X Y /X 0) , n .a . = n ot a va ila bl e; F D = F eu lg en d en si to m et ry , F C M = fl ow cy to m et ry m et ho d; A N = a nt en na , B R = b ra in , H E = ha em oc yt es , M S = m us cl e, O V = o va ri es , S = s pe rm , T S = te st es ; A C = A lli um c ep a (1 C = 1 6. 50 p g) , B O = B os ta ur us ( 1C = 3 .7 0 pg ), BP = B el lis p er en ni s (1 C = 1 .7 6 pg ), D M = D ro so ph ila m el an og as te r (1 C = 0 .1 8 pg ), D V = D ro so ph ila v ir ili s (1 C = 0 .3 4 pg ), G D = G al lu s do m es tic us ( 1C = 1 .2 5 pg ), H S = H om o sa pi en s (1 C = 3 .5 0 pg ), LM = L oc us ta m ig ra to ri a (1 C = 5 .5 0 pg ), M D = M us ca d om es tic a (1 C = 0 .9 0 pg ), M M = M us m us cu lu s (1 C = 3 .3 0 pg ), O M = O nc or hy nc hu s m yk is s (1 C = 2 .6 0 pg ), PA = P er ip la ne ta a m er ic an a (1 C = 3 .4 1 pg ), PS = P is um s at iv um ( 1C = 4 .5 5 pg ), SG = S ch is to ce rc a gr eg ar ia ( 1C = 8 .7 0 pg ). Fa m ily Su bf am ily Sp ec ie s Se x 1C [ pg ] 2n M et ho d C el l T yp e St an da rd S p. R ef er en ce s Su bo rd er : C ae lif er a A cr id id ae A cr id in ae A cr id a co ni ca n. a. 12 .5 5 23 FD H E G D , O M R as ch 1 98 5* A cr id id ae A cr id in ae A cr id a co ni ca M 10 .8 2 23 FD T S G D R ee s et a l. 19 78 A cr id id ae A cr id in ae C al ed ia c ap tiv a M 10 .9 23 FD T S G D R ee s et a l. 19 78 A cr id id ae A cr id in ae C ry pt ob ot hr us c hr ys op ho ru s M 9. 37 23 FD T S G D R ee s et a l. 19 78 A cr id id ae A cr id in ae Sc hi zo bo th ru s fla vo vi tt at us M 7. 5 n. a. FD T S G D R ee s et a l. 19 78 A cr id id ae C at an to pi na e M ac ro to na a us tr al is M 8. 49 23 FD T S G D R ee s et a l. 19 78 A cr id id ae C at an to pi na e Pe ak es ia h os pi ta M 10 .4 7 23 FD T S G D R ee s et a l. 19 78 A cr id id ae C at an to pi na e Ph au la cr id iu m v itt at um M 10 .7 3 23 FD T S G D R ee s et a l. 19 78 A cr id id ae C yr ta ca nt ha cr id in ae Sc hi st oc er ca c an ce lla ta M 9. 49 23 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae C yr ta ca nt ha cr id in ae Sc hi st oc er ca g re ga ri a n. a. 8. 96 23 FD V M M Fo x 19 70 * A cr id id ae C yr ta ca nt ha cr id in ae Sc hi st oc er ca g re ga ri a M 8. 71 23 FD T S M M W ilm or e an d Br ow n 19 75 A cr id id ae C yr ta ca nt ha cr id in ae Sc hi st oc er ca g re ga ri a M 8. 55 23 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae C yr ta ca nt ha cr id in ae Sc hi st oc er ca g re ga ri a M 8. 74 23 FD S n. a. C am ac ho e t a l. 20 15 A cr id id ae C yr ta ca nt ha cr id in ae Sc hi st oc er ca p ar an en si s M 8. 63 23 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae C yr ta ca nt ha cr id in ae Va la ng a ir re gu la ri s M 9. 44 23 FD T S G D R ee s et a l. 19 78 A cr id id ae Ey pr ep oc ne m id in ae Ey pr ep oc ne m is p lo ra ns M 9. 7 23 FD S LM R ui z- R ua no e t a l. 20 11 A cr id id ae Ey pr ep oc ne m id in ae H et er ac ri s ad sp er su s M 6. 34 23 FD T S A C G os al ve z et a l. 19 80 A cr id id ae G om ph oc er in ae G om ph oc er us s ib ir ic us M 8. 95 17 FD T S A C G os al ve z et a l. 19 80 A cr id id ae G om ph oc er in ae C ho rt hi pp us a pi ca lis n. a. 12 .6 1 17 FD T S G D B el da e t a l. 19 91 * A cr id id ae G om ph oc er in ae C ho rt hi pp us b ig ut tu lu s F 11 .3 1 18 FC M M S PS th is s tu dy A cr id id ae G om ph oc er in ae C ho rt hi pp us b in ot at us n. a. 10 .9 1 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae C ho rt hi pp us c f. bi no ta tu s n. a. 10 .3 5 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae C ho rt hi pp us b ru nn eu s M 10 .1 5 17 FD T S A C G os al ve z et a l. 19 80 A cr id id ae G om ph oc er in ae C ho rt hi pp us b ru nn eu s M 9. 46 17 FD T S M M W ilm or e an d Br ow n 19 75 A cr id id ae G om ph oc er in ae C ho rt hi pp us b ru nn eu s M 8. 55 17 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae G om ph oc er in ae C ho rt hi pp us d or sa tu s n. a. 8. 34 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae C ho rt hi pp us d or sa tu s F 12 .0 7 18 FC M M S PS th is s tu dy A cr id id ae G om ph oc er in ae C ho rt hi pp us ja co bs i n. a. 10 .8 4 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae C ho rt hi pp us ju cu nd us n. a. 11 .8 8 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae C ho rt hi pp us lo ng ic or ni s M 8. 58 17 FD T S A C G os al ve z et a l. 19 80 A cr id id ae G om ph oc er in ae C ho rt hi pp us n ev ad en si s n. a. 11 .5 3 17 FD T S G D B el da e t a l. 19 91 114 Martin Husemann et al. Fa m ily Su bf am ily Sp ec ie s Se x 1C [ pg ] 2n M et ho d C el l T yp e St an da rd S p. R ef er en ce s A cr id id ae G om ph oc er in ae Ps eu do ch or th ip pu s pa ra lle lu s n. a. 14 .7 2 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae Ps eu do ch or th ip pu s pa ra lle lu s n. a. 13 .8 3 17 n. a. n. a. n. a. Pe tit pi er re 1 99 6 A cr id id ae G om ph oc er in ae Ps eu do ch or th ip pu s pa ra lle lu s M 13 .3 6 17 FD T S M M W ilm or e an d Br ow n 19 75 A cr id id ae G om ph oc er in ae Ps eu do ch or th ip pu s pa ra lle lu s M 12 .3 1 17 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae G om ph oc er in ae C ho rt hi pp us s ca la ri s n. a. 14 .7 2 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae C ho rt hi pp us v ag an s M 8. 68 17 FD T S A C G os al ve z et a l. 19 80 A cr id id ae G om ph oc er in ae C ho rt hi pp us v ag an s n. a. 8. 64 17 FD T S G D B el da e t a l. 19 91 A cr id id ae G om ph oc er in ae M yr m el eo te tt ix m ac ul at us n. a. 13 .3 8 17 n. a. n. a. n. a. Pe tit pi er re 1 99 6 A cr id id ae G om ph oc er in ae M yr m el eo te tt ix m ac ul at us M 12 .6 6 17 FD T S M M W ilm or e an d Br ow n 19 75 A cr id id ae G om ph oc er in ae M yr m el eo te tt ix m ac ul at us M 12 .1 4 17 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae G om ph oc er in ae O m oc es tu s vi ri du lu s M 13 .1 6 17 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae G om ph oc er in ae St au ro de ru s sc al ar is n. a. 16 .3 4 17 n. a. n. a. n. a. Pe tit pi er re 1 99 6 A cr id id ae M el an op lin ae C am py la ca nt ha o liv ac ea F 6. 98 n. a. FC M BR G D H an ra ha n an d Jo hn st on 2 01 1 A cr id id ae M el an op lin ae C am py la ca nt ha o liv ac ea M 6. 15 n. a. FC M BR G D H an ra ha n an d Jo hn st on 2 01 1 A cr id id ae M el an op lin ae M el an op lu s di ffe re nt ia lis M 6. 79 23 FC M BR PA H an ra ha n an d Jo hn st on 2 01 1 A cr id id ae M el an op lin ae M el an op lu s di ffe re nt ia lis n. a. 6. 23 23 FD H E G D , O M R as ch u np ub l. * A cr id id ae M el an op lin ae M el an op lu s di ffe re nt ia lis n. a. 3. 84 23 FD O V, T S B O Sw ift a nd K le in fe ld 1 95 3* A cr id id ae M el an op lin ae M el an op lu s di ffe re nt ia lis F 7. 26 24 FC M BR PA H an ra ha n an d Jo hn st on 2 01 1 A cr id id ae M el an op lin ae M el an op lu s sa ng ui ni pe s n. a. 5. 83 23 FD H E G D , O M R as ch u np ub l. * A cr id id ae M el an op lin ae Po di sm a pe de st ri s M 16 .9 3 23 /2 4 FD S SG W es te rm an n et a l. 19 87 A cr id id ae O ed ip od in ae A ilo pu s th al as si nu s M 6. 68 23 FD T S G D R ee s et a l. 19 78 A cr id id ae O ed ip od in ae A us tr oi ce te s pu si lla M 6. 29 23 FD T S G D R ee s et a l. 19 78 A cr id id ae O ed ip od in ae G as tr im ar gu s m us ic us M 9. 01 n. a. FD T S G D R ee s et a l. 19 78 A cr id id ae O ed ip od in ae H um be te nu ic or ni s M 8. 21 23 FD T S LM Jo hn a nd H ew itt 1 96 6 A cr id id ae O ed ip od in ae C ho rt oi ce te s te rm in ife ra M 7. 22 23 FD T S M M W ilm or e an d Br ow n 19 75 A cr id id ae O ed ip od in ae C ho rt oi ce te s te rm in ife ra M 5. 99 23 FD T S G D R ee s et a l. 19 78 A cr id id ae O ed ip od in ae Lo cu st a m ig ra to ri a F 6. 44 24 FC M n. a. M M W an g et a l. 20 14 A cr id id ae O ed ip od in ae Lo cu st a m ig ra to ri a n. a. 6. 35 23 FD H E G D , O M R as ch 1 98 5 A cr id id ae O ed ip od in ae Lo cu st a m ig ra to ri a n. a. 6. 27 23 FD V M M Fo x 19 70 A cr id id ae O ed ip od in ae Lo cu st a m ig ra to ri a M 6. 09 23 FD T S M M W ilm or e an d Br ow n 19 75 A cr id id ae O ed ip od in ae Lo cu st a m ig ra to ri a M 5. 47 23 FD T S G D R ee s et a l. 19 78 A cr id id ae O ed ip od in ae Lo cu st a m ig ra to ri a n. a. 5. 28 23 FD S M D Bi er a nd M ül le r 19 69 * A cr id id ae O ed ip od in ae O ed ip od a ca er ul es ce ns F 14 .2 24 FC M M S PS th is s tu dy A cr id id ae O ed ip od in ae Sp hi ng on ot us c ae ru la ns M 12 .5 6 23 FC M M S PS th is s tu dy A cr id id ae O ed ip od in ae Sp hi ng on ot us c ae ru la ns F 13 .3 2 24 FC M M S PS th is s tu dy A cr id id ae O ed ip od in ae St et ho ph ym a gr os su m M 17 .3 6 23 FC M M S PS th is s tu dy A cr id id ae O ed ip od in ae St et ho ph ym a gr os su m F 18 .4 8 24 FC M M S PS th is s tu dy M or ab id ae M or ab in ae W ar ra m ab a vi rg o n. a. 4 15 FD BR G D W hi te a nd W eb b 19 68 115New genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome Fa m ily Su bf am ily Sp ec ie s Se x 1C [ pg ] 2n M et ho d C el l T yp e St an da rd S p. R ef er en ce s M or ab id ae M or ab in ae W ar ra m ab a vi rg o n. a. 3. 75 15 n. a. n. a. n. a. Pe tit pi er re 1 99 6 Su bo rd er : E ns ife ra A no st os to m at id ae D ei na cr id in ae H em id ei na c ra ss id en s 1 M 5. 4 15 FC M A N BP M or ga n- R ic ha rd s 20 05 A no st os to m at id ae D ei na cr id in ae H em id ei na c ra ss id en s 1 F 6. 01 16 FC M A N BP M or ga n- R ic ha rd s 20 05 A no st os to m at id ae D ei na cr id in ae H em id ei na th or ac ic a 1 M 5. 95 15 FC M A N BP M or ga n- R ic ha rd s 20 05 A no st os to m at id ae D ei na cr id in ae H em id ei na th or ac ic a 1 F 6. 53 16 FC M A N BP M or ga n- R ic ha rd s 20 05 G ry lli da e G ry lli na e A ch et a do m es tic us n. a. 2. 38 11 FI A H E D M K os hi ka w a et a l. 20 08 G ry lli da e G ry lli na e A ch et a do m es tic us n. a. 2 11 FD H E G D , O M R as ch 1 98 5 G ry lli da e G ry lli na e A ch et a do m es tic us n. a. 2 11 FD O V, T S M M , H S Li m a- de -F ar ia e t a l. 19 73 G ry lli da e G ry lli na e A ch et a do m es tic us n. a. 2 11 FC M BR D M G re go ry u np ub l. G ry lli da e G ry lli na e A ch et a do m es tic us n. a. 2 11 FI A H E G D G re go ry u np ub l. G ry lli da e G ry lli na e G ry llu s pe nn sy lv an ic us n. a. 2. 68 11 n. a. n. a. n. a. Pe tit pi er re 1 99 6 G ry lli da e G ry lli na e G ry llu s pe nn sy lv an ic us n. a. 2. 06 21 FD S M D Bi er a nd M ül le r 19 69 G ry lli da e G ry lli na e G ry llu s pe nn sy lv an ic us n. a. 2 21 FD H E G D , O M R as ch 1 98 5 G ry lli da e O ec an th in ae O ec an th us n iv eu s n. a. 1. 71 n. a. FC M BR D V H an ra ha n an d Jo hn st on 2 01 1 G ry llo ta lp id ae G ry llo ta lp in ae N eo sc ap te ri sc us b or el lii n. a. 3. 41 n. a. FC M BR G D H an ra ha n an d Jo hn st on 2 01 1 R ha ph id op ho ri da e C eu th op hi lin ae C eu th op hi lu s st yg iu s n. a. 9. 55 n. a. FD H E G D , O M R as ch a nd R as ch 1 98 1 R ha ph id op ho ri da e C eu th op hi lin ae H ad en oe cu s su bt er ra ne us n. a. 1. 55 n. a. FD H E G D , O M R as ch a nd R as ch 1 98 1 Te tt ig on iid ae C on oc ep ha lin ae C on oc ep ha lu s sp . M 2. 65 33 FC M BR G D H an ra ha n an d Jo hn st on 2 01 1 Te tt ig on iid ae C on oc ep ha lin ae C on oc ep ha lu s sp . F 3. 03 34 FC M BR G D H an ra ha n an d Jo hn st on 2 01 1 Te tt ig on iid ae C on oc ep ha lin ae N eo co no ce ph al us tr io ps M 7. 29 n. a. FC M BR G D H an ra ha n an d Jo hn st on 2 01 1 Te tt ig on iid ae C on oc ep ha lin ae N eo co no ce ph al us tr io ps F 7. 93 n. a. FC M BR G D H an ra ha n an d Jo hn st on 2 01 1 Tr id ac ty lid ae n .a . un kn ow n sp . n. a. 2. 63 n. a. FC M BR D V H an ra ha n an d Jo hn st on 2 01 1 Tr ig on iid ae Tr ig on id iin ae La up al a ce ra si na n. a. 1. 93 n. a. FC M BR G D Pe tr ov e t a l. 20 00 116 Martin Husemann et al. the standard in 500 μl of 4°C cold Otto buffer I. The sus- pension of released cells was then filtered through a 42 μm nylon mesh and divided in two parts. One part was stained with 1,000 μl DAPI solution (stock: 25 ml Otto buffer II, 1 ml DAPI (0.1 mg/ml), 25 μl 2-mercaptoetha- nol (2 μl/ml)); the second part was stained with 1,000 μl propidium iodide (PI) solution (stock: 25 ml Otto buff- er II, 1 ml RNase (1 mg/ml), 1 ml PI (1 mg/ml), 25 μl 2-mercaptoethanol) (Doležel et al. 2007). For DAPI analysis, the Partec CyFlow instrument (Partec GmbH, Münster, Germany) with UV LED chip and for PI analysis the Partec SL instrument with a green solid-state laser (Cobolt Samba, 532 nm, 100 mW) were used. Each sample was stained for several minutes before measurement, and 3,500 to 5,000 particles were recorded in each FCM analysis. FCM data were analysed with the Partec FloMax v. 2.52 software (Partec GmbH, Münster, Germany). Combined DAPI and PI measurement results of the same sample express the AT/GC ratio of the genome of the species, the GC content (e.g. Šmarda et al. 2008; Sadílek et al. 2019a, b). The GC content of P. sativum is 38.50% (e.g. Barrow and Meister 2002; Šmarda et al. 2008) and the GC content of the analysed samples was calculated with the Microsoft Excel macro from Šmarda et al. (2008). RESULTS DAPI-stained samples yielded a lower coefficient of variation (CV) than PI-stained samples, on average CV = 2.83% and 3.59% respectively. All the analysed spe- cies of Oedipodinae reached higher genome size values than the analysed species of Gomphocerinae. We were able to measure the genome size of both sexes only in two species (S. caerulans and S. grossum). There, the female/male genome size values clearly reflected the XX/ X0 sex determination system differences. Due to this sex determination system it is generally preferred to report genome size in 2C values rather than the commonly used 1C value. However, to allow for better comparabil- ity, we here report both values. All analysed species of Oedipodinae had distinct genome size (Table 1). The male of S. caerulans had 2C = 25.12 pg (1C = 12.56 pg); the female had 2C = 26.63 pg (1C = 13.32 pg). The female specimen of O. caerulescens exhibited a 2C value of 28.39 pg (1C = 14.20 pg). The largest genome size was recorded in S. grossum, where the male reached 2C = 34.72 pg (1C = 17.36 pg) and the female 2C = 36.95 pg (18.48 pg). Both closely related Gomphocerinae species showed very similar genome siz- es (Table 1): 2C = 22.62 pg (1C = 11.31 pg) in the C. cf. biguttulus female and 2C = 24.14 pg (1C = 12.07) in the female of C. dorsatus. The sample/standard ratio of samples stained with PI was always higher than in DAPI-stained samples of the same specimen, ranging from 11% difference in the female of C. dorsatus to 18% difference in the female of S. grossum. This trend is observable also in the GC content, where C. dorsatus had only 40.82% and the female of S. grossum had 42.35% (Table 1). However, the GC content differences among all species analysed were minimal. DISCUSSION We present new genome size estimates for five spe- cies of Acrididae, one of which represents the largest genome of all insects measured so far, the genome of the female of Stethophyma grossum with 2C = 36.95 pg (1C = 18.48 pg). We also measured a female of C. dorsa- tus with 2C = 24.14 pg (1C = 12.07 pg). This species was measured before using the Feulgen densitometry method with 1C = 8.34 pg (Belda et al. 1991). However, the more recent method of flow cytometry we used is considered more accurate for genome size estimations (e.g. Doležel and Greilhuber 2010). Furthermore, we collected all previous estimates from Gregory (2020) and added few additional resources to provide some basic visualization of the genome size variation in the different subfamilies of Orthoptera (Fig. 1). In total, we gathered 92 (our new data included) estimates of genome sizes belonging to 54 species (Table 2, Fig. 1). These data included 68 estimates for Caelif- era (43 species) and 17 for Ensifera (11 species). They ranged from 1C = 3.75 pg for Warramaba virgo (Key, 1963) (Morabidae) (Petitpierre 1996) to 1C = 18.48 pg for Stethophyma grossum (Oedipodinae, present study) in Caelifera and from 1C = 1.55 pg for Hadenoecus subter- raneus to 1C = 9.55 pg for Ceuthophilus stygius (Scudder, 1861) (both cave Rhaphidophoridae) in Ensifera (Rasch and Rasch 1981). Average 1C values in Ensifera and Caelifera are 3.16 pg (± 2.18 pg) and 9.83 pg (± 3.32 pg) respectively. Further analyses at the family and subfam- ily level are difficult, as most data comes from Acrididae with 66 measurements (78%). The average genome size in Acrididae is 10.01 pg (± 3.19 pg). Within Acrididae, most estimates came from 26 measurements of Gom- phocerinae and 17 of Oedipodinae with average genome sizes of 1C = 11.52 pg (± 2.17 pg) and 9.13 pg (± 4.20 pg) respectively (Table 2, Fig. 1). Generally, the short-horned grasshoppers (Caelifera) appear to have larger genomes compared to the long- 117New genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome horned grasshoppers (Ensifera). However, this is not cor- related with the number of chromosomes. Despite their relatively low male number of chromosomes of 2n = 17 (most of other Acrididae have 2n = 23; e.g. Sylvest- er et al. 2019), Gomphocerinae have some of the largest genome sizes. Their average genome size is 1C = 11.52 pg ranging from 1C = 8.34 pg in C. dorsatus (Belda et al. 1991) to 16.34 pg in Stauroderus scalaris (Petit- pierre 1996; Gregory 2020). Moreover, they show large intraspecific variation in genome size evident from dif- ferent studies (Table 2), for example: 1C = 12.31 pg to 14.72 pg for Pseudochorthippus parallelus (Zetterstedt, 1821) (John and Hewitt 1966; Wilmore and Brown 1975; Belda et al. 1991; Petitpierre 1996) or 1C = 8.55 to 10.15 pg for C. brunneus (John and Hewitt 1966; Wilmore and Brown 1975; Gosalvez et al. 1980). All studies of the two species mentioned above share the method of Feulgen densitometry and used testes to measure genome size. Hence it remains unclear whether this variation is natu- ral or the result of methodological differences. However, it is more likely that the large intraspecific differences are a result of a combination of multiple factors: differ- ent populations analysed, lack of chromosome observa- tions, various standards used and also different instru- mentation could play some role. The variation in genome size is even higher in Oedi- podinae with a minimum of 1C = 5.28 pg for Locusta migratoria (Linnaeus, 1758) (Bier and Müller 1969) and a maximum of 1C = 18.48 pg in Stethophyma grossum. Hence, S. grossum represents the so far largest meas- ured confirmed insect genome. A study by Schielzeth et al. (2014) measured much larger genome sizes for the Gomphocerinae species C. biguttulus with 1C up to 236.05 pg. Due to the enormous variation of the esti- mates in the study and critical methodological issues, Camacho (2016) suggested that these estimates cannot be considered reliable. Hence, we consider our estimate of the S. grossum genome size as the current upper size of insect genomes. Since only very few species have been measured so far, it is expected that this is not the upper bound for genome sizes in grasshoppers or for insects in general. The reasons for the large size of Caelifera genom- es remain largely unknown. However, a recent paper by Shah et al. (2020) suggests that repetitive DNA and especially the expansion of satellite DNA may be a main reason for the large genomes in Orthoptera. The most likely causes are genome duplications at the basis of the Acrididae, which would also explain their specifically high rates in nuclear mitochondrial pseudogenes (numts, Figure 1. Relative fluorescence histograms for samples stained with PI. 2C peaks represent diploid cells and 4C peaks represent cells in the G2 phase of the cell cycle. with replicated DNA. Standard used: P. sativum 2C = 9.09 pg. (A) S. grossum female with 2C = 36.95 pg. (B) C. biguttulus female with 2C = 22.62 pg. 118 Martin Husemann et al. Bensasson et al. 2000; Song et al. 2008) posing difficul- ties to species identification using DNA barcoding and to phylogenetic reconstruction (Hawlitschek et al. 2017, Song et al. 2018). It may also explain why only a sin- gle incomplete genome is available to date (Wang et al. 2014). Grasshopper genome sizes remain a major obsta- cle to genomic research, and many further studies will be required to understand genome size variation and evolution in Orthoptera. ACKNOWLEDGEMENT We thank Torsten Demuth for providing locality access and help with sampling. We also thank Martin Fikáček (Charles University, Prague, Czech Republic) for financial support for processing the samples in FCM laboratory of Tomáš Urfus (Charles University, Prague, Czech Republic) from the botany department. DATA AVAILABILITY STATEMENT All data generated and used in this article is includ- ed as tables and figures. GEOLOCATION INFORMATION All sampling for this study was performed 2019 in Hamburg, Georgswerder (Germany, 53.5097°N 10.0301°E). REFERENCES Barrow M, Meister A. 2002. Lack of correlation between AT frequency and genome size in higher plants and the effect of non-randomness of base sequences on dye binding. Cytometry 47:1–7. Belda JE, Cabrero J, Camacho JPM, Rufas JS. 1991. Role of C-heterochromatin in variation of nuclear DNA amount in the genus Chorthippus (Orthoptera, Acrididae). Cytobios 67:13–21. Bensasson D, Zhang D-X, Hewitt GM. 2000. Frequent assimilation of mitochondrial DANN by grasshopper nuclear genomes. Mol Biol Evol 17:406-415. Bier K, Müller W. 1969. DNA-Messungen bei Insekten und eine Hypothese über retardierte Evolution und besonderen DNA-Reichtum in Tierreich. Biol Zen- tralblatt 88:425–449. Camacho JPM, Ruiz-Ruano FJ, Martin-Blázquez R, Cabrero J, Lorite P, Cabral-de-Mello DC, Bakkali M. 2015. A step to the gigantic genome of the desert Figure 2. Genome Size variation in the different subfamilies of Orthoptera visualized as a boxplot. Provided is the number of measure- ments (N) and the number of species (sp) these measurements were derived of (some of the species were measured repeatedly by differ- ent authors). Most of the data excerpted from database Gregory (2020) completed with another original data comprehended in Table 2. *unknown species genome size was analysed, determined only on family level. 119New genome size estimates for band-winged and slant-faced grasshoppers reveal the so far largest measured insect genome locust: chromosome sizes and repeated DNAs. Chro- mosoma 124:263–275. Camacho JPM. 2016. Comment on Schielzeth et al. (2014): “Genome size variation affects song attrac- tiveness in grasshoppers: Evidence for sexual selec- tion against large genomes“. Evolution 70:1428–1430. Doležel J, Geilhuber J. 2010. Nuclear genome size: are we getting closer? Cytometry Part A 77A:635–642. Doležel J, Greilhuber J, Lucretti S, Meister A, Lysák MA, Nardi L, Obermayer R. 1998. Plant genome size esti- mation by flow cytometry: Inter-laboratory compari- son. Ann Bot 82:17–26. Doležel J, Greilhuber J, Suda J. 2007. Estimation of nucle- ar DNA content in plants using flow cytometry. Nat Prot 2:2233–2244. Fox DP. 1970. A non-doubling DNA series in somatic tis- sues of the locusts Schistocerca gregaria (Forskål) and Locusta migratoria (Linn.). Chromosoma 29:446–461. Gosalvez J, López-Fernandez C, Esponda P. 1980. Varia- bility of the DNA content in five orthopteran species. Caryologia 33:275–281. Gregory TR. 2020. Animal genome size. Database 2020. http://www.genomesize.com Accessed 19 April 2020. Hanrahan SJ, Johnston JS 2011. New genome size esti- mates of 134 species of arthropods. Chrom Res 19:809–823. Hawlitschek O, Morinère J, Lehmann GUC, Lehmann AW, Kropf M, Dunz A, Glaw F, Detcharoen M, Schmidt S, Hausmann A, Szucsich NU, Caetano- Wyler SA, Haszprunar G. 2017. DNA barcoding of crickets, katydids and grasshoppers (Orthoptera) from Central Europe with focus in Austria, Germany and Switzerland. Mol Ecol Res 17:1037–1053. John B, Hewitt GM. 1966. Karyotype stability and DNA variability in the Acrididae. Chromosoma 20:155–172. Koshikawa S, Miyazaki S, Cornette R, Matsumoto T, Miura T. 2008. Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae). Naturwissenschaften 95:859–867. Li F, Zhao X, Li M, He K, Huang C, Zhou Y, Li Z, Wal- ters JR. 2019. Insect genomes: Progress and challeng- es. Insect Mol Biol 28:739-758. Lima-de-Faria A, Gustafsson T, Jaworska H. 1973. Ampli- fication of ribosomal DNA in Acheta. II. The num- ber of nucleotide pairs of the chromosomes and chromomeres involved in amplification. Hereditas 73:119–142. Morgan-Richards M. 2005. Chromosome rearrangements are not accompanied by expected genome size chang- es in tree weta Hemideina thoracica (Orthoptera, Anostostomatidae) J Orthop Res 14:143-148. Pellicer J, Fay MF, Leitch IJ. 2010. The largest eukaryotic genome of them all? Bot J Linn Soc 164:10-15. Petitpierre E. 1996. Molecular cytogenetics and taxonomy of insects, with particular reference to the Coleop- tera. Int J Insect Morph Embry 25:115–133. Petrov DA, Sangster TA, Johnston JS, Hartl DL, Shaw KL. 2000. Evidence for DNA loss as a determinant of genome size. Science 287:1060–1062 Rasch EM. 1985. DNA “standards” and the range of accu- rate DNA estimates by Feulgen absorption micro- spectrophotometry In: Advances in Microscopy. Cowden RR, Harrison SH (Eds.). Alan R. Liss, New York. 137–166. Rasch EM, Rasch RW. 1981. Cytophotometric determina- tion of genome size for two species of cave crickets (Orthoptera, Rhaphidophoridae). J Histochem Cyto- chem 29:885. Rees H, Shaw DD, Wilkinson P. 1978. Nuclear DNA variation among acridid grasshoppers. Proc Roy Soc London B 202:517–525. Robinson GE, Hackett KJ, Purcell-Miramontes M, Brown SJ, Evans JD, Goldsmith MR, Lawson D, Okamuro J, Robertson HM, Schneider DJ. 2011. Creating a buzz about insect genomes. Science 331:1386. Rodríguez A, Burgon JD, Lyra M, Irisarri I, Baurain D, Blaustein L, Göcmen B, Künzel S, Mable BK, Nolte AW, Veith M, Steinfartz S, Elmer KR, Philippe H, Vences M. 2017. Inferring the shallow phylogeny of true salamanders (Salamandra) by multiple phylog- enomic approaches. Mol Phyl Evol 115:16–26. Ruiz-Ruano FJ, Ruiz-Estévez M, Rodríguez-Pérez J, López-Pino JL, Cabrero J, Camacho JPM. 2011. DNA Amount of X and B Chromosomes in the Grasshop- pers Eyprepocnemis plorans and Locusta migratoria. Cytogenet Genome Res 134:120–126. Sadílek D, Urfus T, Hadrava J, Vilímová J, Suda J. 2019a. Nuclear genome size in contrast to sex chromosome number variability in the human bed bug, Cimex lectularius (Heteroptera: Cimicidae). Cytometry Part A 95A:746–756. Sadílek D, Urfus T, Vilímová J. 2019b. Genome size and sex chromosome variability of bed bugs feeding on animal hosts compared to Cimex lectularius parasitiz- ing human (Heteroptera: Cimicidae). Cytometry Part A 95A:1158–1166. Schielzeth H, Streitner C, Lampe U, Franzke A, Reinhold K. 2014. Genome size variation affects song attrac- tiveness in grasshoppers: Evidence for sexual selec- tion against large genomes. Evolution 68:3629–3635. Shah A, Hoffman JI, Schielzeth H. 2020. Comparative analysis of genomic repeat content in Gomphocerine grasshoppers reveals expansions of satellite DNA and 120 Martin Husemann et al. helitrons in species with unusually large genomes. Genome Biol Evol 12:1180-1193. Šmarda P, Bureš P, Horová L, Foggi B, Rossi G. 2008. Genome size and GC content evolution of Festuca: ancestral expansion and subsequent reduction. Ann Bot 101:421–433. Song H, Mariño-Pérez R, Woller DA, Cigliano MM. 2018. Evolution, diversification, and biogeography of grasshoppers (Orthoptera: Acrididae). Insect Syst Div 2:3;1-25. Swift H, Kleinfeld R. 1953. DNA in grasshopper spermat- ogenesis, oögenesis, and cleavage. Phys Zool 26:301– 311. Sylvester T, Blackmon H. 2019. Idiosycratic patterns of chromosome evolution are the rule not the excep- tion. https://evobir.shinyapps.io/PolyneopteraDB/ Current version of the database is 0.1 last updated 12 August 2019. Wang X, Fang X, Yang P, Jiang X, Jiang F, Zhao D, Li B, Cui F, Wei J, Ma C, Wang Y, He J, Luo Y, Wang Z, Guo X, Guo W, Wang X, Zhang Y, Yang M, Hao S, Chen B, Ma Z, Yu D, Xiong Z, Zhu Y, Fan D, Han L, Wang B, Chen Y, Wang J. 2014. The locust genome provides insight into swarm formation and long-dis- tance flight. Nat Comm 5:2957. Westerman M, Barton NH, Hewitt GM. 1987. Differences in DNA content between two chromosomal races of the grasshopper Podisma pedestris. Heredity 58:221– 228. White MJD, Webb GC. 1968. Origin and evolution of parthenogenetic reproduction in the grasshopper Moraba virgo (Eumastacidae: Morabinae). Aust J Zool 16:647–671. Wilmore PJ, Brown AK. 1975. Molecular properties of Orthopteran DNA. Chromosoma 51:337–345.