Unusual chromosome polymorphism and heterochromatin variation in the Argentinean population of the necrophagous fly Lucilia sericata (Diptera: Calliphoridae), comparison with other populations and evolutionary aspects

Heterochromatin may vary qualitatively, quantitatively, and in its location both in and between species. There were differences recorded in sex chromosomes, satellite sizes and location of C-bands in specimens of Lucilia sericata (Meigen, 1826) from three localities in the Buenos Aires region, Argentina (Bernal, Castelar, and Buenos Aires City). Mitotic analyses revealed the same diploid chromosome number of 2n = 10 + XY / XX (male / female) and the same size, morphology, and C-banding patterns on their autosomes. However, there are different morphotypes of X and Y chromosomes with a distinctive heterochromatin content in both arms. Four morphotypes of X (X1, X2, X3, and X4) and three of Y (Y1, Y2, and Y3) were recorded. The X metacentric and Y subtelocentric chromosomes were identifi ed based on their very large and small sizes, respectively, and both had large heterochromatic blocks. These intraspecifi c differences are mainly due to quantitative variations in non-coding repetitive sequences located in the arms of both X and Y sex chromosomes. The specimens were very polymorphic both between and within the three localities sampled. Cytogenetic studies on specimens of L. sericata from the Nearctic, Palaearctic and Ethiopian regions also report differences in the morphology of the sex chromosomes and their C-banding patterns, some of which may have evolved independently. Our cytogenetic observations on L. sericata indicate that the accumulation of heterochromatin in the genome could be involved in chromosomal divergence and karyotype evolution of this species, as demonstrated in other Diptera. The polymorphic sex chromosomes are therefore important for understanding the evolution within species as well as speciation.


INTRODUCTION
The cosmopolitan and geographically widespread blowfl ies, the Calliphoridae, are a group of great medical, forensic, and veterinary signifi cance (Greenberg, 1991;Byrd & Castner, 2001;Opletalová et al., 2012). Lucilia sericata (Meigen, 1826) is one of the fi rst insects to arrive at a corpse (Centeno et al., 2002;Ames & Turner, 2003) and, therefore, is the primary and most accurate indicator of the time of death (Centeno et al., 2002).
Based on morphological differences, 32 species of Lucilia are described (Stevens & Wall, 1996;Whitworth, 2010Whitworth, , 2014, with 23 species occurring in the Neotropical Region (South America, Central America, the West Indies and portions of southern Mexico along the east and west coasts).
To determine the karyotype, we measured at least twenty wellspread mitotic metaphases for each female and male larva of L. sericata from CABA, Bernal, and Castelar. The total chromo-es in the constitutive heterochromatin in several isolated populations of different species are reported (White, 1973;Baimai, 1998;Rafael & Tadei, 2000;Selivon et al., 2005;Ullerich & Schöttke, 2006;Cáceres et al., 2009;Agrawal et al., 2010;Acuña-Morera et al., 2011;Chirino et al., 2015). Moreover, DNA studies on L. sericata from North America detected that there are three subpopulations there based on AFLP genotype analyses (Picard & Wells, 2010) whereas a random mating occurs in Eastern European fl ies and there are no subpopulations based on the chaetotaxy of the thorax and eight microsatellite loci (Diakova et al., 2018). Thus, the population genetic structure and morphological features of L. sericata may depend on geographic origin and associated differences in life-history traits. The processes of genetic differentiation may include chromosomal rearrangements, leading to diverse chromosome morphologies and variation in inter-and intra-specifi c constitutive heterochromatin, resulting in quantitative changes in repetitive DNA sequences (Grewal & Jia, 2007).
The karyotype and C-banding pattern of L. sericata from Argentina are reported in a previous study (Chirino et al., 2015). Here, we determine the C-banding on the autosomes and sex chromosomes in specimens of L. sericata from three localities in the Buenos Aires region (Fig. 1) because the X chromosomes had two distinctive morphologies ( Fig. 1c in Chirino et al., 2015). We measured their relative lengths, size of satellites, centromeric index, arm ratio, and distribution and percentage of heterochromatin.

Insects
Females of Lucilia sericata from three localities in the Buenos Aires region, Argentina ( Fig. 1) were collected using beef meat as bait, between October 2016 and May 2017. Flies were sampled in some length measurements (TCL; mean ± SE) of all bivalents and sex chromosomes, relative average length (μm; length of an autosomal bivalent as a percentage of the TCL) and relative chromosomal percentage (%) were obtained using MicroMeasure software for Windows, version 3.3 (Reeves & Tear, 2000). Measurements were of chromosomes at metaphase. Differences in the lengths of chromosomes of males and females from the three localities were analysed using Kruskal-Wallis ANOVAs on ranks for global comparisons (P < 0.05), followed by Kruskal-Wallis all-pairwise comparison test for contrasts between treatments because the data were not normally distributed and did not show homoscedasticity. Statistical analyses were performed using In-foStat software (Di Rienzo et al., 2015). The constitutive heterochromatin percentage as the C-positive area divided by the TCL was calculated. The C-banded region in the short and long arms of pairs II and III and in both sex chromosomes was calculated as the ratio between the C-positive area and the arm length. The heterochromatic blocks in the satellites of the X and Y chromosome were determined as the C-positive area of the satellite concerning its total length. Idiograms illustrating the C-band patterns were based on the results obtained from several individuals.

Microscopy and image processing
Preparations were observed under a Leica DM2000 epifl uorescence microscope equipped with a Leica DFC345 FX CCD camera and Leica Application Suite (LAS), version 3.8 (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). Blackand-white images of chromosomes were recorded, pseudo-coloured and processed with Adobe Photoshop CS6 Version 6.1 software.  Morphotypes of sex chromosomes and patterns on the 2 nd and 3 rd chromosomal pairs. A − female X 1 X 1 , DD, Bb, Bernal; B − female X 1 X 2 , DD, BB, CABA; C − female X 1 X 4 , SS, Bb, CABA; D − female X 3 X 4 , DS, Bb, CABA; E − female X 4 X 4 , SS, bb, Castelar; F − male X 4 Y 1 , SS, Bb, Castelar; G − male X 1 Y 2 , SS, BB, Bernal; H − male X 4 Y 3 , SS, Bb, CABA. Heteromorphic sex pair in detail (A, C-D). Very large Cpositive blocks are observed in the pairs of autosomes II and III, and both X and Y sex chromosomes. X, Y -sex chromosomes. Arrows indicate the double C+ heterochromatin band (DD) near the secondary constriction on chromosome 2. Arrowheads show the single C+ heterochromatin band (SS) near the secondary constriction on chromosome 2. Open arrowheads indicate the heterozygous C-band pattern (DS) on the secondary constriction on chromosome 2. Double lines indicate the C-block pattern (BB) on the secondary constriction on chromosome 3. Single lines indicate the C-band pattern (bb) on the secondary constriction on chromosome 3. Asterisks indicate the presence of one C-block (B) and one C-band (b), heterozygous state (Bb), on the secondary constriction on chromosome 3. Bar = 10 μm.

RESULTS
The autosomes from specimens collected from the three localities (CABA, Bernal, and Castelar) were very similar in morphology and size (H = 2.49, P = 0.2884) and gradually decreased in size (H = 153.95, P < 0.0001; Table 1). Four types of X (X 1 , X 2 , X 3 , X 4 ) and three types of Y chromosomes (Y 1 , Y 2 , Y 3 ) were distinguished, characterized by differences in the location of the secondary constriction, the size of their satellites and C-banding patterns (Figs 2, 3; Tables 1, 2). The X was larger than the Y chromosome (H = 216.91, P < 0.0001). No statistical differences in sizes were detected between the four X chromosomes or between the three Ys of individuals from the same or different localities. Data for all specimens are shown together (Figs 2, 3; Tables 1, 2). Centromeric C-bands were found on all chromosomes. The variation in the amount of constitutive heterochromatin in the pericentromeric regions (bands and blocks) of autosome pairs II and III and X and Y chromosomes are shown in Figs 2 and 3 and Table 2.
In specimens from all three sites, the autosome pair II had one or two well-defi ned interstitial C-bands on the short arm associated with the secondary constriction. According to the number of these interstitial C-bands, there were three different patterns in females and males: homozygous for the presence of two C-bands on each short arm (double banding pattern = DD) ( Fig. 2A, B), homozygous for the presence of one C-band on each short arm (single banding pattern = SS) (Fig. 2C, E-H) and heterozygous for the presence of one C-band (S) on the short arm of one homologue and two C-bands (D) on the short arm of the other ( Fig. 2D; Table 3). The fi rst two C-banding patterns were predominant in specimens from all three sites, while only one female from each locality showed the DS pattern (Table 3). Furthermore, we observed differences in the size of the heterochromatic blocks around the secondary constriction on the short arms of autosome pair III in both females and males from all three sites. This autosome pair can exhibit one C-block on each short arm (block pattern = BB) (Fig. 2B, G), one C-band on each short arm (band pattern = bb) (Fig. 2E) or it can be heteromorphic for the size of the band, i.e., the C-band has a different size in each homologue (Bb; Fig. 2A, C, D, F, H). The BB and Bb patterns were common in both sexes from all three sites, whereas the bb pattern was rare and not detected in specimens from CABA (Table 3).
Sex chromosomes showed greater variation in the content and distribution of heterochromatic blocks than the autosomes (Figs 2, 3; Table 2). The X 1 and X 2 chromosomes were very similar in size, relation of arms, small size of their satellites, and percentage of heterochromatin. The X 1 chromosome differed from X 2 in the content and distribution of the heterochromatic block, with about 75% of the X 1 satellite but only about 50% of the X 2 satellite heterochromatic (Figs 2B, 3). The X 3 and X 4 chromosomes had larger satellites than X 1 and X 2 . The satellite of X 3 was mainly euchromatic (Fig. 2D), with only a small terminal area of heterochromatin, whereas the satellite of X 4 was mainly heterochromatic (Fig. 2C-F, H). The morphotypes of Y also differed in the relative content of heterochromatin Morphology of chromosomes. m -metacentric, m-sm -metacentric-submetacentric, sm -submetacentric, st -subtelocentric. * Statistical comparisons were analysed using Kruskal-Wallis tests. Different letters indicate signifi cant differences in post-hoc analyses (P < 0.05). ** Distance of the secondary constriction concerning the centromere in the short arm of pairs II, III, and X chromosomes, and in the long arm of Y chromosomes. "-" -lack secondary constriction. 38.54 -78.81 -* Differences in C-positive heterochromatin percentage in the total length (TL) of the centromeric and pericentromeric areas of each pair of chromosomes. "-" -lack heterochromatin. ** C-positive heterochromatin percentage in short arm of pairs II and III, and in the heterochromatic blocks in short and long arms of sex chromosomes (including each satellite). # C-positive heterochromatin percentage exclusively recorded in the satellites of X and Y chromosomes.
in the satellite. The Y 1 chromosome had the largest fully heterochromatic satellite (Fig. 2F); Y 2 had a medium-sized heterochromatic block covering nearly 50% of its satellite (Fig. 2G), and Y 3 had an euchromatic satellite (Fig. 2H). The polymorphism of sex chromosomes varied both within and between collecting sites. However, the X 1 and Y 1 chromosomes were the commonest morphotypes recorded at all the sites studied (Table 4). Specimens from Bernal were only of the X 1 morphotype (X 1 X 1 in females, X 1 Y 1 or X 1 Y 2 in males). Samples from Castelar were of all four X morphotypes; with females of four cytotypes (X 1 X 4 , X 2 X 4 , X 3 X 4 , X 4 X 4 ), with the X 4 being present in all plates examined, males were of two Y morphotypes (Y 1 and Y 2 ), with Y 2 being less common. Females from CABA were also of all four X morphotypes in several combinations, whereas males displayed only of the X 1 , X 3 and X 4 (with a predominance of the latter) and a high proportion of the Y 3 morphotype, which was only detected at this locality.

DISCUSSION
This study revealed a surprisingly high chromosome variability in terms of the amount, distribution, and location of the constitutive heterochromatin in the pericentromeric regions of autosome pairs II and III, and in the X and Y chromosomes of L. sericata from the three sites sampled in Argentina. There is remarkable conservation of autosomal banding patterns, in contrast to the variety of X and Y chromosomes in specimens from all three sites, including four metacentric and three subtelocentric morphologies, with different distributions of pericentromeric heterochromatic blocks. Polymorphisms involving heterochromatin are frequently found in other dipteran insects, mammals, and plants (Baimai, 1998;Rafael & Tadei, 2000;Rudra & Bahadur, 2013;Steiner et al., 2014;Siljak-Yakovlev et al., 2017). However, it is not common to fi nd heterochromatic polymorphisms in the same population, as in L. sericata from Argentina. The small number of females used in this study undoubtedly infl uenced our results and underestimated or skewed the real cytogenetic pattern. Adults arriving together at baits could be related and indicate a higher level of inbreeding than would be expected from a random sample, although for the three Argentinean sites sampled for L. sericata there is no evidence of a geographic barrier to gene fl ow, isolation by distance or a population genetic structure, as has been detected in the United States and Europe (Picard & Wells, 2010;Diakova et al., 2018). Therefore, it is diffi cult to interpret the recorded polymorphism in terms of population genetics (genetic drift, differential selection, migration, etc.).
Cytogenetic studies on L. sericata from several biogeographical regions have revealed intraspecifi c variability in the morphologies and sizes of X and Y, in the extent of the C segments they contain and that the sex chromosomes are more likely to rearrangements by the accumulation of constitutive heterochromatin than the autosomes (Boyes, 1961;El-Bassiony, 2006;Ullerich & Schöttke, 2006;Acuña-Morera et al., 2011;Chirino et al., 2015;this study). Concerning the content and distribution of heterochromatic blocks on the sex chromosomes, only the Argentinean population is known to have different morphotypes of X and Y sex chromosomes. The X chromosomes of individuals from Argentina, Colombia, and Egypt are  (629) Abbreviations: F -female, M -male, DD -double banding pattern, SS -single banding pattern, DS -heterozygous for the presence of one C-band D (S) and two C-bands (D), BB -C-block pattern, bb -C-band pattern, Bb -heterozygous for the presence of one C-block (B) and one C-band (b). * Number of adults (and cells) examined of L. sericata for each locality and sex. Kingdom are telocentric. The long and short arms of the Y chromosome in Colombian populations are heterochromatic and euchromatic, respectively, whereas those in German populations are heterochromatic, but lack a secondary constriction as in the Argentinean population. Therefore, the chromosomes of L. sericata blowfl ies from different regions differ in their C-banding patterns. The processes of genetic differentiation include chromosomal rearrangements giving rise to diverse chromosome morphologies and variation in intra-specifi c constitutive heterochromatin, which results in quantitative changes of repetitive DNA sequences that lead to karyotype evolution (Baimai, 1998;Rafael & Tadei, 2000;He et al., 2003;this study). In L. sericata, the sex chromosomes are very variable in the content and distribution of heterochromatic blocks compared with the autosomes. An intra-specifi c variation in sex chromosomes through the accumulation of constitutive heterochromatin is a common phenomenon in reproductively isolated populations of other widely distributed species of Diptera (Baimai, 1998;Rafael & Tadei, 2000;He et al., 2003). In addition, chromosome polymorphisms were also detected in the three Argentinean population studied and in one Uruguayan population of Hematobia irritans (Linnaeus, 1758) (Diptera: Muscidae) (Forneris et al., 2015). In this species, there are seven karyotypes with different frequencies in populations, which also differ in chromosome banding, chromosome rearrangements (polymorphisms), and chromosome number. Therefore, qualitative and quantitative variations in heterochromatinization associated with changes in its amount and location both within and between species, appear to be an important step in the morphological differentiation of simple sex chromosome systems.
The karyotype data described herein demonstrate that L. sericata is undergoing chromosomal differentiation and karyotype evolution through the accumulation of heterochromatin in the genome. Genetic differentiation in several populations of a species, such as described here, could lead to evolutionary divergence at the chromosomal level and consequently, to speciation. Cryptic and sibling species, or incipient speciation, are frequently detected fi rst at the cytological level in species of Diptera (Baimai, 1998;He et al., 2003). Future studies using a greater number of females could reveal the evolutionary mechanisms behind the observed cytogenetic differentiation. Karyological studies may also help elucidate the high plasticity of genomes at the chromosomal level, potentially leading to adap tation and speciation.