Research Article |
Corresponding author: Mara Garcia Tavares ( mtavares@ufv.br ) Academic editor: Dorota Lachowska
© 2018 Alexandra Avelar Silva, Lucas Soares Braga, Alberto Soares Corrêa, Valerie Renee Holmes, John Spencer Johnston, Brenda Oppert, Raul Narciso Carvalho Guedes, Mara Garcia Tavares.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Silva AA, Braga LS, Corrêa AS, Holmes VR, Johnston JS, Oppert B, Guedes RNC, Tavares MG (2018) Comparative cytogenetics and derived phylogenic relationship among Sitophilus grain weevils (Coleoptera, Curculionidae, Dryophthorinae). Comparative Cytogenetics 12(2): 223-245. https://doi.org/10.3897/CompCytogen.v12i2.26412
|
Cytogenetic characteristics and genome size are powerful tools for species characterization and identification of cryptic species, providing critical insights into phylogenetic and evolutionary relationships. Sitophilus Linnaeus, 1758 grain weevils can benefit from such tools as key pest species of stored products and also as sources of archeological information on human history and past urban environments. Moreover, the phylogenetic relationship among these weevil species remains controversial and is largely based on single DNA fragment analyses. Therefore, cytogenetic analyses and genome size determinations were performed for four Sitophilus grain weevil species, namely the granary weevil Sitophilus granarius (Linnaeus, 1758), the tamarind weevil S. linearis (Herbst, 1797), the rice weevil S. oryzae (Linnaeus, 1763), and the maize weevil S. zeamais Motschulsky, 1855. Both maize and rice weevils exhibited the same chromosome number (2n=22; 10 A + Xyp). In contrast, the granary and tamarind weevils exhibited higher chromosome number (2n=24; 11 A + Xyp and 11 A + neo-XY, respectively). The nuclear DNA content of these species was not proportionally related to either chromosome number or heterochromatin amount. Maize and rice weevils exhibited similar and larger genome sizes (0.730±0.003 pg and 0.786±0.003 pg, respectively), followed by the granary weevil (0.553±0.003 pg), and the tamarind weevil (0.440±0.001 pg). Parsimony phylogenetic analysis of the insect karyotypes indicate that S. zeamais and S. oryzae were phylogenetically closer than S. granarius and S. linearis, which were more closely related and share a more recent ancestral relationship.
karyotypes, C-banding, fluorochromes, heterochromatin, stored products, evolutionary history
Closely related species usually exhibit similar karyotypes concerning chromosome number and morphology. However, other characteristics such as the amount, size and distribution of heterochromatic blocks and/or nucleolus organizing regions (NORs) can vary considerably, even among cryptic species, which makes cytogenetic analyses powerful tools for species characterization and identification (
Interspecific divergence is also associated with chromosome variation (
Genome size is another trait useful in comparative studies in a variety of taxonomic levels (
Curiously, cytogenetic studies are non-existent for several taxa and species groups that have recognized importance as pest species, and exhibit archaeological relevance, such as grain weevils of the genus Sitophilus Linnaeus, 1758 (
The genus Sitophilus comprises fourteen species, three of which (the rice weevil S. oryzae (Linnaeus, 1763), the maize weevil S. zeamais and the granary weevil S. granarius (Linnaeus, 1758)), are of greater scientific interest because of their broadly recognized status as primary pest species of stored products throughout the world (
The phylogenetic relationship among these weevils is controversial (
The aims of this study were to: 1) perform a comparative cytogenetic characterization among S. granarius, S. linearis, S. oryzae and S. zeamais); 2) quantify the genome size of these four species; and 3) perform a more complete karyotype-based phylogenetic analysis with these species. The data will contribute to the understanding of the genomic organization and the taxonomic status of these species.
Sitophilus granarius were obtained from wheat kernels in Manhattan (Kansas, USA; 39°11'18"N; 96°36'21"W); S. linearis was obtained from tamarind seeds in Piracicaba (São Paulo, Brazil; 22°43'31"S; 47°38'57"W) and Montes Claros (Minas Gerais, Brazil; 16°44'06"S; 43°51'42"W); and S. oryzae was obtained from rice kernels in Cascavel (Paraná, Brazil; 24°57'21"S; 53°27'19"W) and São Borja (Rio Grande do Sul; Brazil; 28°39'38"S; 56°00'16"W). Samples of S. zeamais were obtained from maize kernels in Cruzeiro do Sul (Acre, Brazil; 07°37'52"S; 72°40'12"W) and Porto Alegre (Rio Grande do Sul, Brazil; 30°01'59"S; 51°13'48"W).
The last larval instars of each weevil species (i.e., Sitophilus granarius, S. linearis, S. oryzae and S. zeamais) were used for karyotyping and adult insects were used for genome size determination. Insects of each species were reared in glass containers (0.5 L) in an environmentally controlled rearing room (18 ± 2 °C, 70 ± 10% relative humidity and a photoperiod of 12:12 h L:D), containing grains of either wheat (S. granarius), tamarind fruits (S. linearis) or maize grains (S. oryzae and S. zeamais). The larvae were extracted from their respective hosts after inspection of different substrate grains with a LX-60 specimen radiography system equipped with a 14-bit digital camera (Faxitron X-Ray Corp., Wheeling, IL, USA). The adults were sieved from the grains, snap-frozen in dry ice and maintained under –80 °C until genome size determination.
The cerebral ganglia of individuals of the last larval stage were processed according to
Mapping of ribosomal DNA was performed with probes for 18S rDNA obtained by PCR amplification using primers F (5’ TCATATGCTTGTCTAAAGA-3’) and R (3’-TCTAATTTTTTCAAAGTAAACGC-5’) designed for Melipona quinquefasciata Lepeletier, 1836 (
The sex chromosomes were identified by comparing female and male karyotypes. Ten male karyotypes of each species were mounted in order to establish which chromosomes do not form an exact pair. These chromosomes were considered the sex ones and, by comparison, it was possible to establish the chromosomes corresponding to the sex pair, in females. The sex determination system of the four species, in turn, was recognized by analysing meiotic figures from the testes following
An average of 20 metaphases per slide were evaluated with an Olympus BX60 microscope coupled to an image capturing system (Image-Pro Plus Version 6.3, Media Cybernetics 2009). The slides stained with fluorochromes (CMA3/DAPI) were analyzed with an epifluorescence light microscope using excitation filters WB (λ = 330–385 nm) and WU (λ = 450–480 nm) under oil immersion at 100× magnification. The chromosomes were classified according to
Genome size was estimated by flow cytometry as described in
The relationship among the four species of Sitophilus grain weevils was determined using a matrix with a total of 20 karyotype characters, where five characters were parsimony informative (exhibiting at least two characters distinct among operation taxonomic units [OTUs]; i.e., the weevil species studied) (Table
Sitophilus granarius:
The karyotype of S. granarius showed 2n=24 chromosomes, including 11 pairs of autosomes and a pair of sex chromosomes. Most autosomal pairs, except pairs 1, 4 and 5, exhibited a metacentric morphology. The first autosomal pair was longer than the remaining and the other pairs gradually decrease in size. The submetacentric X chromosome was similar in size to the 11th chromosome pair, while the metacentric Y chromosome was the smallest element in the set (Figures
Karyotypes of Sitophilus granarius (a), S. linearis (b), S. oryzae (c) and S. zeamais (d). The first and the second lines for each species represent female karyotypes stained with Giemsa and C-banding, respectively, while the third line represents male karyotypes stained with Giemsa (a, b, c) or C-band (d). Bar = 5 μm.
Sequential staining with fluorochromes, in turn, allowed the identification of CMA3+ regions only in the centromere of the sixth autosomal pair and in one of the Y arms, whereas DAPI stained the short arm of the X chromosome and the complementary arm of the Y chromosome (Fig.
Metaphases of Sitophilus granarius (a–d), S. linearis (e–h), S. oryzae (i–k) and S. zeamais (l–n) stained with CMA3 and DAPI or submitted to rDNA 18S FISH. Pictures a, b, d, e, f, h represent male cells, while the remaining ones are from females. The arrows indicate the rDNA location, while blank and solid arrowheads indicate the X and the y chromosomes, respectively. Bar = 5 μm.
The analysis of male meiotic cells revealed a sex chromosome system of the Xyp type (Fig.
Meiotic male metaphase cells of Sitophilus granarius (a), S. linearis (b), S. oryzae (c) and S. zeamais (d), stained with Giemsa, showing the typical parachute association of the sex chromosomes (arrowhead) in all species, except in S. linearis. The asterisks indicate a B chromosome. Bar = 5 μm.
Sitophilus linearis:
The karyotype of this species also exhibited 2n=24 chromosomes, which gradually decrease in size. Most autosomal chromosomes were metacentric, except pairs 1, 2, 10 and 11, which were submetacentric. The submetacentric X chromosome was the longest element in the karyotype, while the Y showed a subtelocentric morphology equal in size to one of the medium-sized chromosomes (Fig.
The chromosomal mapping of major rDNA clusters (18S) confirmed that ribosomal genes were located in the telomeric region of pair 10 and in the short arm of the Y chromosome. So, with both CMA3 and FISH, females showed two positive signals, while males showed three positive signals (Fig.
The typical parachute association of the sex chromosomes present in S. granarius was not observed, despite the analysis of several metaphase I cells. Instead, analysis of these cells showed an XY association in all cells evaluated (Fig.
Sitophilus oryzae:
This species exhibited a karyotype consisting of 2n=22 chromosomes that gradually decreased in size. Nine autosomal pairs showed a metacentric morphology; only the autosomal pair 6 was submetacentric (Fig.
Observation of meiotic cells indicated the sex pair exhibiting a parachute configuration, as in S. granarius. Therefore, its meioformulae were n=10 + XX and n=10 + Xyp, for females and males, respectively (Fig.
Sitophilus zeamais:
As described by
Autosomes and the X chromosome exhibited small heterochromatic blocks in the centromeric region after C-banding and DAPI staining, while the Y chromosome was entirely euchromatic (Figures
Analysis of meiotic cells confirmed that the sex pair exhibited the parachute configuration, as in S. granarius and S. oryzae. Therefore, their meioformulae were n=10 + XX and 10 + Xyp, for females and males respectively (Fig.
The mean genome size (1C) estimates for the four Sitophilus species analysed in the present study and their chromosome numbers are in Table
Genome size estimates for the grain weevils Sitophilus granarius, S. linearis, S. oryzae and S. zeamais; the number of individuals analyzed (N) and chromosome number are indicated.
Species | Haploid genome size pg ± SE(Mbp ± SE)Female (F) Male (M) | N (F/M) | Chromosome number | |
---|---|---|---|---|
Sitophilus granarius | 0.5533 ± 0.003 (541.1 ± 2.9) | 0.5561 ± 0.003 (543.9 ± 3.0) | 5/4 | 2n=24 |
Sitophilus linearis | 0.4395 ± 0.001 (429.8 ± 0.6) | 0.4351 ± 0.001 (425.5 ± 1.4) | 2/4 | 2n=24 |
Sitophilus oryzae | 0.7865 ± 0.002 (769.2 ± 1.9) | 0.7852 ± 0.003 (768.0 ± 3.1) | 4/6 | 2n=22 |
Sitophilus zeamais | 0.7296 ± 0.008 (713.5 ± 7.5) | 0.7252 ± 0.003 (709.2 ± 2.8)0.7860 ± 0.006 (768.7 ± 5.7) | 5/3-/2 | 2n=222n=22 + Bs |
Matrix data of karyotype features of the Sitophilus pest species and outgroup Otiorhynchus bisulcatus (Coleoptera: Curculionidae).
Karyotype features | Species | ||||
---|---|---|---|---|---|
S. zeamais | S. oryzae | S. granarius | S. linearis | O. bisulcatus* | |
Number of chromosomes | 0 | 0 | 1 | 1 | 0 |
Presence of B chromosomes | 1 | 0 | 0 | 0 | 0 |
Sex-chromosome system (Xyp) | 1 | 1 | 1 | 0 | 1 |
22 metacentric chromosomes | 1 | 0 | 0 | 0 | 0 |
20 metacentric chromosomes | 0 | 1 | 0 | 0 | 0 |
18 metacentric chromosomes | 0 | 0 | 0 | 1 | 0 |
16 metacentric chromosomes | 0 | 0 | 1 | 0 | 1 |
0 submetacentric chromosomes | 1 | 0 | 0 | 0 | 0 |
2 submetacentric chromosomes | 0 | 1 | 0 | 0 | 0 |
8 submetacentric chromosomes | 0 | 0 | 1 | 1 | 0 |
6 submetacentric chromosomes | 0 | 0 | 0 | 1 | 0 |
4 submetacentric chromosomes | 0 | 0 | 0 | 0 | 1 |
1 telocentric chromosome | 0 | 0 | 0 | 1 | 0 |
Number of the sexual pair | 0 | 1 | 2 | 3 | ? |
Morphology of the X chromosome | 1 | 1 | 0 | 0 | 1 |
Morphology of the y chromosome | 0 | 1 | 1 | 2 | 0 |
Banda C pattern | 0 | 0 | 1 | 0 | 0 |
DAPI distribution | 0 | 0 | 1 | 0 | 1 |
CMA3 distribution** | 0 | 1 | 2 | 3 | 4 |
NOR localization (FISH)** | 0 | 1 | 2 | 3 | 4 |
The phylogenetic analysis showed that S. zeamais and S. oryzae were phylogenetically closer than S. granarius and S. linearis, supported for the clade with bootstrap = 66 (Table
The chromosome number of 2n=22, the parachute configuration, and the prevalence of metacentric chromosomes that we found in S. oryzae and S. zeamais represent cytogenetic characteristics already described in most species of Curculionidae surveyed so far (
First, the higher number of chromosomes observed in S. linearis and S. granarius (2n =24) suggests that the karyotype of these species may have evolved by centric fission of autosomes. Alternatively, the karyotypes of S. oryzae and S. zeamais, that have 2n=22 chromosomes, could have originated as a result of pericentric inversions in small pairs followed by fusions between them. The first scenario, however, seems more probable, once 2n=22 is the prevalent and seems to be the ancestral chromosomal number for Curculionidae species (
Secondly, cytogenetic analysis revealed differences among the four species related to the morphology and size of sex chromosomes. For example, in S. granarius and S. linearis, the X chromosome was submetacentric, but the Y chromosome was metacentric and subtelocentric, respectively. In contrast, S. oryzae and S. zeamais exhibited metacentric X chromosomes, but whereas the Y chromosome in S. zeamais was punctiform, that of S. oryzae was metacentric and not so small as in S. zeamais. In S. linearis, in particular, the X chromosome represents the longest element in the karyotype and the Y is also significantly longer than the four/five small autosomes pairs. They are also much larger than the sexual ones in the other three species analysed. Additionally, B chromosomes were found exclusively in some populations of S. zeamais. Together, these characteristics facilitate the identification of this particular species.
Thirdly, as the sex chromosomes of S. linearis are large and form a well differentiated figure from the Xyp of the other Sitophilus species in first meiosis, we propose that this species has a sex determination system of the neo-XY type. However, translocation(s) between an autosomal pair and the sex chromosomes in an ancestral species, with increase of the X-Y sizes and reduction in the number of autosomes, does not seem to explain the origin of the neo-XY system in S. linearis. Although the(se) translocation(s) were already observe in some insect species (
A more plausible explanation for the neo-XY system in S. linearis would be the contributions of more than one autosomal pair to form the large neo-XY chromosomes, with decreases in their sizes, but without reduction in their number, as reported for Calcosoma atlas (Dutrillaux and Dutrillaux 2013). In this sense, cytogenetic analysis provided clear evidence of the absence of the first larger autosome pair in the karyotype S. linearis, a characteristic easily recognized in the other three Sitophilus species and, consequently, its participation in this process. Additionally, considering the actual size of the sex chromosomes of S. linearis, the fact that the two/three first pairs of chromosomes of this species are more similar in size than the equivalent chromosomes in the karyotypes of other Sitophilus species, and the diminutive size of the sexual chromosomes of its phylogenetically closer species, S. granarius (see below), we can suggest that these chromosomes could also be involved in the formation of the neo-XY chromosomes of S. linearis, with small reductions in their sizes. The presence of rDNA clusters in the Y chromosomes of S. linearis, as discussed above, is another indication of these translocations. However, further studies will be necessary to confirm this mechanism, the autosomal pairs involved in the process and the exact chromosomal rearrangements concerning the evolution of the neo-sex chromosomes of S. linearis.
The genus Sitophilus, especially S. granarius, possesses a small amount of heterochromatin that was located preferentially at the centromeric region, as in most Curculionidae (
The coincidence of DAPI staining with the C-banding marks in the chromosomes of S. granarius, S. linearis and S. oryzae, as well as in S. zeamais (
The analysis of the localization and distribution of rRNA clusters largely contributed toward the cytogenetic characterization of the four Sitophilus species analysed. The findings indicate that ribosomal genes are located in a single autosomal pair in three (S. granarius, S. oryzae and S. zeamais) of the four analysed species (different pairs for each species). This corroborates previous reports suggesting that an autosome pair performs as a nucleolus organizer in Coleoptera (
In S. linearis, however, positive CMA3 and FISH stainings were also detected in the Y chromosome. Data obtained, therefore, evidenced that in this species, the Y chromosome also bears rDNA clusters. To our knowledge, this is the first time that rDNA genes is mapped directly (FISH) on the Y chromosome in Curculionidae, while the presence of rDNA genes on the X or on both sex chromosome (besides autosomes ones) have already been documented in some species of Coleoptera, by FISH analysis (
Additionally, CMA3 and FISH results revealed fluorescent labels in only one of the homologous of the pair 3 in S. zeamais. Although methodological problems cannot be excluded as a source of this variability, it seems unlikely that both techniques would yield the same results, even because they were efficient for the detection of the localization of rDNA genes in the other three Sitophilus species. Thus, we believe that this represents a size polymorphism between these homologous and, consequently, that both of them would contain rDNA genes, but that in one of them, the low copy number of ribosomal cistrons (< 10kb [Yiang and Gill 1994]) could not be detected with the probe used here. This suggestion is supported by the fact that this result was found in both populations analysed (Cruzeiro do Sul and Porto Alegre).
The flow cytometry analyses provided a preliminary scenario about the haploid genome size variation among the Sitophilus species. The genome size of S. oryzae (0.7865 pg) was similar to S. zeamais (0.7296 pg), whereas S. granarius (0.5533 pg) exhibited a small genome size, and an even smaller was found in S. linearis (0.4395 pg). These findings also corroborate the reportedly high intra genus variation in arthropods, as S. oryzae has 66% more DNA than S. linearis. Although genome size variation is mainly due to variation in the amount of non-coding DNA not necessarily reflecting phylogenetic relationship, this does not seem the case for grain weevils, as we reported here. The variation in DNA content among these four weevil species is consistent and reinforces the phylogenetic relationship among them based on the karyotypes reported here and also on their endosymbionts (
Cytometry data also provided evidence that nuclear DNA content is not proportionally related to either the chromosomal number, or the heterochromatin amount in Sitophilus species. In the first case, both smaller genome species (i.e., S. linearis and S. granarius) exhibit higher chromosome numbers than the species with higher genome sizes (S. oryzae and S. zeamais). In the second case, S. linearis exhibited a similar amount of heterochromatin to both S. oryzae and S. zeamais, and a larger amount than S. granarius, despite the smaller genome size of S. linearis. The genome sizes of Sitophilus males and females were similar, although three species exhibit the Xyp system, while the tamarind weevil exhibits the neo-XY sex determination system. This findings are suggestive that the genome size variation observed in Sitophilus grain weevils may be a result of repetitive DNA sequences (e.g., satellite DNA, transposable elements etc.) accounting for a more complex gene regulation in species with larger genome size, as reported for eukaryotes (Comeron 2006,
The obtained genome size of the Sitophilus species were within the previously described range for eight other species of Curculionidae, that include four of the genus Anthonomus Germar, 1817 (0.62-0.86 pg – Bárcenas-Ortega 2005,
Worth noting is also the fact that two genome size estimates were obtained for S. zeamais males. Considering that this species may possess from 0-4 B chromosomes, their presence in some individuals explain the difference observed. However, we were unable to carry out cytogenetic and flow cytometry analyses using the same individuals. Consequently, we could neither establish the number of B chromosomes that different individuals possessed nor the contribution of each B chromosome to the whole genome.
Finally, the parsimony phylogenetic analysis had only mild bootstrap support due to the limited number of informative karyotype characters available, but it does agree with the descriptive analysis of Sitophilus karyotype, which provides evidence that S. zeamais and S. oryzae are phylogenetically closer when compared with S. granarius and S. linearis. The new finding not previously reported is the higher proximity of S. granarius to S. linearis, suggesting a common and more recent ancestry for both species. This finding is also consistent with the genome size and the number of chromosomes of these species, the closer association of the granary weevil with stored grains losing its flight ability (
The ancient origin (ca. 8.7 million years ago) and closer association between the maize and rice weevils were recently reinforced with comprehensive molecular data (
The ancient origin of the grain weevils, likely pre-dating the onset of agriculture in Southeast Asia and the India subcontinent, together with their recent adaptation to stored products, make these earlier invader species useful for tracking grain and trade routes since the Neolithic period between 15,200 and 2,000 BC (
In summary, we were able to describe the karyotype of the tamarind weevil and extend the karyotypic analysis of the maize weevil, allowing a comparative cytogenetic characterization of the four Sitophilus grain weevils (S. granarius, S. linearis, S. oryzae, and S. zeamais). A more complete karyotype-based phylogenetic analysis of these four species, aided by the quantification of genome size in each, shed light on the conflicting phylogeny of the grain weevil species. The ancestral and closer phylogenetic association between S. zeamais and S. oryzae was recognized, as was the more recent cluster encompassing S. granarius and S. linearis and a shared ancestral relationship.
We are grateful to Dr. Bh. Subramayan for providing specimens of S. granarius for our study, and to Dr. Silvia G. Pompolo, Dr. Denilce M. Lopes and Dr. Lucio Antonio O. Campos for their technical assistance and valuable suggestions. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. We are also grateful to the National Council of Scientific and Technological Development (CNPq, Brazilian Ministry of Science and Technology), CAPES Foundation (Brazilian Ministry of Education), the Minas Gerais State Foundation for Research Aid (FAPEMIG) and Arthur Bernardes Foundation (FUNARBE) for the financial support provided.