Loss of Y chromosome may be a synapomorphy of the tribe Lepturini ( Coleoptera : Cerambycidae

Phylogeny and higher classifi cation of the cerambycid subfamily Lepturinae remain controversial. Here we report the results of a cytogenetic study of 18 species currently classifi ed in Lepturini and 12 species in other tribes of Lepturinae (1 in Oxymirini, 1 in Rhamnusiini and 10 in Rhagiini) from Western Europe. The male sex chromosome formula is XY in all Rhagiini, Oxymirini and in Grammoptera rufi cornis in the Lepturini (whose tribal placement may be doubtful), and X0 in all the remaining Lepturini. The rarity of the X0 formula in other Cerambycidae indicates that the Y chromosome was lost in a common ancestor of the Lepturini or a subgroup thereof, indicating its monophyletic origin. The number of chromosomes is stable in the Lepturini that lack a Y chromosome (19,X/20,XX in males and females, respectively), but varies from 20 to 24 in the remaining genera, probably the consequence of evolution by chromosome fi ssion. Whereas all the males with 19 or 20 chromosomes have an early gametogenesis, which is achieved before the imago stage, the species with more than 20 chromosomes seem to have a delayed male gametogenesis, which is still active in the young imagoes. The species of Rhagiini with 22 chromosomes may constitute a monophyletic group. * Corresponding author; e-mail: bdutrill@mnhn.fr INTRODUCTION Lepturinae constitute one of the large subfamilies of Cerambycidae, with more than 1000 species distributed mainly in the Northern hemisphere. Their systematic classifi cation remains controversial; Bousquet et al. (2009) list 8 tribes: Desmocerini Blanchard, 1845, Encyclopini LeConte, 1873, Lepturini Latreille, 1802, Oxymirini Danilevsky, 1997, Rhagiini Kirby, 1837, Rhamnusiini Sama, 2009, Teledapini Pascoe, 1871 and Xylosteini Reitter, 1913, and the Necydalinae Lacordaire, 1825 is considered a separate subfamily. According to Svacha & Lawrence (2014), lepturine classifi cation is unsatisfactory, unstable and obviously non-phylogenetic. In Western Europe, only 5 of the above lepturine tribes are present, among which, Rhagiini and Lepturini are by far the most numerous, with at least 13 and 23 genera, respectively (Löbl & Smetana, 2010). Surprisingly, chromosome studies of Lepturinae are very scarce and almost restricted to the pioneer studies of Teppner (1966, 1968). These old reports show that the karyotypes of the few species studied are composed of 20 or 22 chromosomes, numbers shared by most other Cerambycidae and other Polyphaga (Smith & Virkki, 1978). Most of the specimens of Lepturini they studied are females, with an XX sex-chromosome formula and the very Eur. J. Entomol. 115: 45–52, 2018 doi: 10.14411/eje.2018.006


INTRODUCTION
Lepturinae constitute one of the large subfamilies of Cerambycidae, with more than 1000 species distributed mainly in the Northern hemisphere.Their systematic classifi cation remains controversial; Bousquet et al. (2009) list 8 tribes: Desmocerini Blanchard, 1845, Encyclopini LeConte, 1873, Lepturini Latreille, 1802, Oxymirini Danilevsky, 1997, Rhagiini Kirby, 1837, Rhamnusiini Sama, 2009, Teledapini Pascoe, 1871and Xylosteini Reitter, 1913, and the Necydalinae Lacordaire, 1825 is considered a separate subfamily.According to Svacha & Lawrence (2014), lepturine classifi cation is unsatisfactory, unstable and obviously non-phylogenetic.In Western Europe, only 5 of the above lepturine tribes are present, among which, Rhagiini and Lepturini are by far the most numerous, with at least 13 and 23 genera, respectively (Löbl & Smetana, 2010).Surprisingly, chromosome studies of Lepturinae are very scarce and almost restricted to the pioneer studies of Teppner (1966Teppner ( , 1968)).These old reports show that the karyotypes of the few species studied are composed of 20 or 22 chromosomes, numbers shared by most other Cerambycidae and other Polyphaga (Smith & Virkki, 1978).Most of the specimens of Lepturini they studied are females, with an XX sex-chromosome formula and the very Carnoy I fi xative (ethanol : acetic acid, 3 : 1), in which the cells were suspended and left for at least 30 min.After one change of fi xative, the cells were spread on wet and cold slides or conserved in the tube for a few days at 6°C before use.Slides were stained with Giemsa, photographed and C-banded according to Angus (1982).This technique was used for both adults and larvae.

Testicular cells
The same method was applied, except for the treatment with 0.88 KCl, which was reduced from 1 h to 10 min.In most species, cell divisions were obtained only in larvae and pupae.

Eggs
They were gently cleaned and transferred into a micro-centrifuge tube containing 0.5 to 1 ml of a 0.88 M KCl solution with added colcemid and squashed using a piston, as above.The rest of the technique is as for the mid-gut, but only diluted calf serum was used for the hypotonic shock.These techniques are derived from those of Dutrillaux &Chevin (1968) andAngus (1982).

RESULTS
The chromosome numbers, sex chromosome formulae and morphology of the X chromosome of the 29 species studied are summarized in Tables 2 and 3, and karyotypes are shown in Figs 1-4.The scarce data in the literature are also included in the tables.
we show that all the males of species currently classifi ed in Lepturini (Grammoptera rufi cornis excepted) have an X0 sex formula, while all the males of Rhagiini and Oxymirini (plus G. rufi cornis) have an XY sex formula.

Insects
Most of the species studied were collected in France.They are listed in Table 1, with collecting place, stage of development and the tissues studied.Nomenclature used is that of Löbl & Smetana (2010).

Mid-gut cells
After anaesthesia using ethyl acetate, the abdomen was removed and placed in a drop of an aqueous solution of 0.88 M KCl.The mid-gut was extracted, placed in 1 ml of the same solution with the addition of colcemid and dissected to obtain a cell suspension.After 1 h, this suspension was transferred to a microcentrifuge tube (VWR International SAS, Strasbourg, France) and centrifuged (5 min at about 800 g).After elimination of the supernatant, the pellet was suspended in 0.5 ml of a hypotonic solution (either a 0.55 M KCl solution or foetal calf serum diluted 1:3 in distilled water), where they were squashed in the tube and suspended using a piston (VWR) adjusted to the internal diameter of the tube.The volume was increased to 1.5 ml.After 10 min, the cell suspension was centrifuged.The supernatant was replaced by

Chromosomes
All the males have an XY and the 3 females studied have an XX sex formula.The X chromosome may be either acrocentric (P.lurida), or sub-metacentric (B.interrogationis), or metacentric (D. collaris).Its size is in the range of the smallest autosomes and the Y is punctiform.Thus, the sex chromosomes of species belonging to this tribe look like those of most other cerambycid beetles and may be plesiomorphic.The chromosome number of 20, recorded for the genera Rhagium Fabricius, 1775, Brachyta Fairmaire, 1864, Pachyta Dejean, 1821 and Pidonia Mulsant, 1863 is also quite standard in Cerambycidae, as in Polyphaga in general.
The karyotypes of 6 species, 3 from the literature and 3 from this study, belonging to the genera Acmaeops Le-Conte, 1850, Gaurotes LeConte, 1850 and Dinoptera Mulsant, 1863 comprise 22 chromosomes.The sizes of the autosomes of the species with 22 chromosomes (G.virginea, D. collaris and A. pratensis, Fig. 1b-e) progressively decrease without a size gap.In contrast, in P. quadrimaculata, P. lurida and B. interrogationis, there is a size discontinuity, with chromosome 1 much larger than the others.In species of the genus Rhagium, the size discontinuity is between chromosomes 2 and 3 (Fig. 1k and l).
It proved very diffi cult to obtain C-banding, as frequently it was almost absent, very faint, or limited to the centromere region of a few autosomes.The X was C-banded in only a few species and the Y was always very pale (Fig. 1e, g, i, j and l).In spite of these diffi culties, the variations in heterochromatin and chromosome morphology were suffi cient to reveal that all the karyotypes differed from each other.

Gametogenesis
Although probably mostly young adults were captured (in May at low altitudes and June or early July above 1600 m.), all the males with 20 chromosomes had completed their gametogenesis and only bouquets of spermatozoa were occasionally found in the testes.In contrast, we found that gametogenesis was still active in an adult of G. virginea (2n = 22), which provided us with 10 + Xy p metaphases I.

Chromosomes
All the females studied by us have a 20, XX karyotype, in agreement with the data in the earlier literature (Tables 2 and 3).Almost all the males (15 of 16 species) have a 19, X karyotype, which differs from the data of Teppner (1968) who proposes 20 chromosomes for the males of A. dubia and "Leptura ochracea" (a synonym of Anastrangalia reyi).The karyotypes of the species of Lepturini look fairly similar after Giemsa staining, with 9 pairs of metacentric or sub-metacentric autosomes.The karyotype of R. maculata (Figs 2h,3h), however, differs with one pair of acrocentric autosomes.The X is most frequently sub-metacentric.In all these species, pair N°1 is much larger than pair N°2, as in some species of Rhagiini.The karyotype of G. rufi cornis clearly differs from that of other Lepturini: it is composed of 24 chromosomes of regularly decreasing size and its male sex formula is XY.This karyotype is shown in Fig. 1f, g, near those of species of Rhagiini with 22 chromosomes (Fig. 1b-e), which facilitates their comparison and displays their resemblance.
C-banding was also hard to obtain and generally faint and not systematized.It was present in the region of the centromere of either all chromosomes, as in S. melanura This study Smith, 1953 This study This study This study Ehara, 1956Ehara, 1956 This study This study Teppner, 1968 This study This study Teppner, 1968Teppner, 1968 This (Fig. 3l), or only some chromosomes, and even lacking in many species (Fig. 3d-k).C-bands were occasionally present at telomeres (A.dubia and S. rubra, Fig. 3d and f) or in interstitial regions (S. stragulata and P. cerambyciformis, Fig. 3e and k).The centromere region of the X chromosome could be either C-banded as in A. dubia (Fig. 3d), or not, as in L. aurulenta (Fig. 3g).

Gametogenesis
In all but one species studied, male gametogenesis had ceased and only bouquets of spermatozoa were occasionally found in the testes.The exception was G. rufi cornis, in which spermatocytes I at diakinesis were still present in the testicles of the adult.Their meioformula is 11 + Xy p .

Oxymirini
(Table 2, Fig. 4) The karyotype of O. cursor is composed of 18 autosomes with 2 pairs that are acrocentric.With a size gap between pairs 2 and 3, it generally looks like that of the genus Rhagium.The very large size of the sex chromosomes is however quite unusual.Their parallel and tightly associated chromatids indicate a heterochromatic composition, which is confi rmed by their intense C-banding, especially on the Y chromosome.

Rhamnusiini (Table 2)
Only one female larva of R. bicolor was studied; its karyo type was 2n = 20 and XX sex chromosomes.

Chromosome Y loss may characterize Lepturini amongst Lepturinae
The karyotypes of Rhagiini and Lepturini do not differ very much.Most of the females have 20 chromosomes and the males 19 or 20 chromosomes.The decrease from 20 to 19 is the consequence of the lack of chromosome Y, which occurs in the Lepturini, but not in the other tribes studied.In other Cerambycid beetles, the lack of chromosome Y is rare: 1/105 species calculated from the Smith & Virkki (1978) compilation and 1/104 non-Lepturini species in our own unpublished data.Thus, the males of Lepturinae, as those of Cerambycidae in general, originally had a Y chromosome, which was conserved in Rhagiini and Oxymirini, Table 3. Data on the sex of the specimens of Lepturini studied and their main chromosomal characters.Sex chrom.-sex chromosome formula; X morph.-morphology of chromosome X; A -acrocentric; M -metacentric; SM -sub-metacentric; 2n -chromosome number.Data from literature included. 1-given as Leptura ochracea in Teppner (1968).and lost in all or most Lepturini.The two non-Lepturinae species of Cerambycidae reported to lack chromosome Y, S. barbatum (Manna & Lahiri, 1972) and O. amputator (Dutrillaux & Dutrillaux, 2008), belong to the Cerambycinae and Lamiinae, respectively.Thus, the known losses of chromosome Y are the consequence of at least 3 independent events, which occurred in 3 different subfamilies of Cerambycidae.Two of these events seem to be limited to a single or a few species and may be recent.The loss affecting Lepturini may be more ancient, because it is recorded in almost all the species studied and in a total of 17 species and 11 genera.These 11 genera make up about half of those present in Western Europe, but only a small proportion of the world fauna, which is composed of about 120 genera.Indeed, more studies are necessary to determine if the species studied here are representative of all Lepturini or if  they constitute a clade inside Lepturini.The case of G. ruficornis remains ambiguous.The genus Grammoptera Audinet-Serville, 1835 was alternatively classifi ed in Rhagiini or Lepturini, but the most recent classifi cations favour the Lepturini hypothesis.The presence of a Y chromosome in the male would support the Rhagiini hypothesis, also proposed on the basis of the larvae (Svacha & Danilevsky, 1989), but is not in agreement with the molecular data of Sýkorová (2008), which places Grammoptera (represented by two species) in Lepturini and far from the monophyletic group of Rhagiini with 22 chromosomes."Grammoptera" in the molecular tree of Saito & Saito (2003) is actually Alosterna chalybeella (Bates, 1884), and the sequence is moreover misidentifi ed (P.Švácha, pers.comm.).The multiple nomenclatural changes at the levels of genera and tribes of Lepturinae indicate that objective criteria are lacking (Svacha & Lawrence, 2014), and indeed, cytogenetic data could help in their systematic classifi cation.

Species of Lepturini
As discussed elsewhere (Dutrillaux & Dutrillaux, 2017), the presence or absence of chromosome Y seems to have no consequences for the phenotype of the males in beetles: only the balance X/XX determines the sex.Chromosome Y appears to be dispensable, with a role limited to the facilitation of the migration of the X at meiosis through the Xy p structure.However, in X0 males, other strategies involving nucleolar proteins compensate for the lack of chromosome Y (Dutrillaux & Dutrillaux, 2017).At metaphase I, the unique X remains embedded in nucleolar proteins and forms a structure, which was probably interpreted as an Xy p in earlier publications.If, as generally accepted, the males of ancestors of Polyphaga had a Y chromosome, the evolutionary trend is its loss.The example of Lepturini, in which the lack of the Y chromosome was completely overlooked, suggests that the occurrence of X0 males may remain undiscovered in other beetle families.This was the case for Cantharidae, in which James & Angus (2007) found X0 males in species previously described as having an Xy p structure at meiosis.

The deviations in chromosome numbers may defi ne monophyletic taxa
Considering both the literature (Smith & Virkki, 1978) and our own data, it appears that the karyotype of a group of species of Rhagiini belonging to the genera Acmaeops, Gaurotes and Dinoptera is composed of 22, and not the 20 chromosomes present in most Cerambycidae (and presumably plesiomorphic).In addition, in species with 22 chromosomes, the size of the autosomes progressively decreases, whereas both in the remaining Rhagiini and Lepturini, there are discontinuities in chromosome sizes.The karyotypes with 22 chromosomes may have been derived from a karyotype with 20 chromosomes by chromosome fi ssion, possibly involving chromosome 1, which is very large in karyotypes with 20 chromosomes.This is in good agreement with molecular data where the genera with 22 chromosomes, together with some other probably related genera whose karyotypes are unknown (Pseudogaurotina, Lemula, Brachysomida, Rhondia), form monophyletic groups (Saito & Saito, 2003;Sýkorová, 2008).The in-crease to 24 chromosomes of gradually decreasing size in G. rufi cornis may or may not be related to those genera of Rhagiini with 22 chromosomes.Interestingly, the increase in chromosome number may be associated with a developmental particularity: a delayed gametogenesis.In Lepturinae, male gametogenesis ends at the pupal stage (Ehara, 1951;Dutrillaux, 2008).This probably explains the scarcity of cytogenetic data on this subfamily, considering that most of the earlier studies on beetles were done using the gonads of adults.We confi rmed the early gametogenesis in most of the species used in this study, with the exception of Gaurotes virginea and Grammoptera rufi cornis in which gametogenesis occurs in young adults.The literature survey (Tables 2 and 3) indicates that cytogenetic results on dividing meiotic cells are reported for 5 species belonging to 3 genera: Rhagium bifasciatum, R. inquisitor, Acmaeops proteus, Gaurotes doris and G. suvorovi.The data on the 2 species of Rhagium, were obtained from pre-imaginal stages, confi rming their early gametogenesis (Teppner, 1968).In contrast, the data for the two species of Gaurotes were obtained from the imago (Ehara, 1956).The information is not given for A. proteus.These 3 last species have 22 chromosomes.No other results on meiotic chromosomes were found for male Lepturinae, and we repeatedly failed to obtain proliferating germ cells in the testicles of the young adults we studied.As a working hypothesis, we propose that those species with 22-24 chromosomes may be also distinguished by their relatively late gametogenesis.
In conclusion, this study of the male sex chromosomes of 11 species of Lepturinae from Western Europe currently classifi ed in the "Rhagiini" (this tribe may not be monophyletic) are of the classical XY type, whereas those of 17/18 species (the exception is Grammoptera rufi cornis, which may be misclassifi ed) of Lepturini are X0.Further studies, however are needed to confi rm that this loss of chromosome Y characterizes all the species of Lepturini, which could constitute an apomorphy of this tribe whose defi nition remains controversial.In Rhagiini, a group of species with more than 20 chromosomes and late gametogenesis apparently constitutes a monophyletic group, which is in agreement with the limited available molecular data (Saito & Saito, 2003;Sýkorová, 2008).With its very large sex chromosomes, the karyotype of O. cursor (Oxymirini) is unusual, but their enlargement is due to heterochromatin amplifi cation, a rapid process, which may have little meaning for phylogenetic studies.The 2n = 20 female karyotype of Rhamnusium bicolor (Rhamnusiini) may further support the presumption that this should be the plesiomorphic chromosome number.

Fig. 4 .
Fig. 4. Giemsa stained (a) and C-banded (b) karyotypes of male Oxymirus cursor.The very large size of the X and Y chromosomes is due to heterochromatin amplifi cation, which is fully C-banded on the Y and partially C-banded on the X.

Table 1 .
Species of Lepturinae included in the cytogenetic study, with collection data (material from France if not stated otherwise), the stage of development and the organ or tissue successfully analysed.

Table 2 .
Sex and main chromosomal data of the specimens of Rhagiini, Oxymirini and Rhamnusiini studied.Se x chrom.-sex chromosome formula; X morph.-morphology of chromosome X; A -acrocentric; M -metacentric; SM -sub-metacentric; h+ -heterochromatin amplifi cation; 2n -diploid chromosome number.Data from literature included. 1-only the chromosome number is given for the female.