Research Article |
Corresponding author: Peter H. Adler ( padler@clemson.edu ) Academic editor: Paraskeva Michailova
© 2016 Peter H. Adler, Alparslan Yildirim, Zuhal Onder, G. Taskin Tasci, Onder Duzlu, M. Ozkan Arslan, Arif Ciloglu, Baris Sari, Nilgun Parmaksizoglu, Abdullah Inci.
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:
Adler PH, Yildirim A, Onder Z, G. Tasci T, Duzlu O, Arslan MO, Ciloglu A, Sari B, Parmaksizoglu N, Inci A (2016) Rearrangement hotspots in the sex chromosome of the Palearctic black fly Simulium bergi (Diptera, Simuliidae). Comparative Cytogenetics 10(2): 295-310. https://doi.org/10.3897/CompCytogen.v10i2.8855
|
An extreme example of nonrandom rearrangements, especially inversion breaks, is described in the polytene chromosomes of the black fly Simulium bergi Rubtsov, 1956 from Armenia and Turkey. A total of 48 rearrangements was discovered, relative to the standard banding sequence for the subgenus Simulium Latreille, 1802. One rearrangement, an inversion (IIS-C) in the short arm of the second chromosome, was fixed. Six (12.5%) of the rearrangements were autosomal polymorphisms, and the remaining 41 (85.4%) were sex linked. More than 40 X- and Y-linked rearrangements, predominantly inversions, were clustered in the long arm of the second chromosome (IIL), representing about 15% of the total complement. The pattern conforms to a nonrandom model of chromosome breakage, perhaps associated with an underlying molecular mechanism.
Caucasus Mountains, nonrandom breakage, polytene chromosomes, sex linkage
The sex chromosomes of the Simuliidae have commanded attention because of their suggested role in driving speciation (
Unlike the separate heteromorphic X and Y chromosomes of organisms such as Anopheles Meigen, 1818 mosquitoes and Drosophila Fallén, 1823, any one of the three chromosomes (I, II, or III) functions as both the X and the Y chromosome in the Simuliidae. The simuliid sex-chromosome system, therefore, is more similar to that in the closely related family Chironomidae in which the sex chromosomes are generally undifferentiated, although males are sometimes distinguished by rearrangements, such as inversions, that mark the Y chromosome (
Inversions often build on one another to produce elaborate sex chromosomes in a particular arm of the Simuliidae (
Simulium (Simulium) bergi Rubtsov, 1956, a black fly in the S. venustum species group (
General features of the karyotype of S. bergi from Armenia have been provided, such as the lengths of the polytene chromosomes (
Larvae (penultimate and ultimate instars) were collected with forceps primarily from trailing vegetation in one stream each in Armenia and Turkey (Table
Collection sites for larvae of Simulium bergi used in chromosomal analyses.
Country | Location | Latitude Longitude | Elevation (m asl) | Collection Date | Females: Males |
---|---|---|---|---|---|
ARMENIA | Shirak Province, Saragyugh | 41°08.51'N, 43°50.05'E | ca. 2150 | 14 June 2002 | 5:3 |
TURKEY | Kars Province, Bogatepe | 40°48.37'N, 42°53.37'E | ca. 2200 | 07 June 2015 | 13:17 |
The posterior portion of each larval abdomen was removed and processed for Feulgen staining, following procedures of
The chromosomal banding sequences of all stained larvae were compared with maps of the standard reference sequence for the subgenus Simulium. For this comparison, we used the standard maps of
We identified the sex chromosome of S. bergi, based on a preponderance of one or more chromosomal rearrangements in one sex. We then followed the banding sequence of the homologue with the sex-linked rearrangement(s) to determine if additional rearrangements were on the same (cis) or a different (trans) homologue. In the heterogametic sex—the male (XY)—determination of linkage was complicated by heterozygosity. Thus, twisting and overlapping homologues sometimes could not be followed adequately to determine if two rearrangements were cis or trans; in these cases, X or Y linkage could not be resolved. Because females were the homogametic sex (XX), we inferred that any rearrangement in the IIL arm of females was X linked.
Stained and unstained portions of two Armenian larvae (and two pupae) and some larvae from Turkey were transferred to 80% ethanol and deposited in the Clemson University Arthropod Collection, South Carolina, USA, along with all photographic negatives of chromosomes. The majority of Armenian and Turkish larvae were placed in the Department of Parasitology, Erciyes University, Turkey, for future molecular analysis.
General features. The banding sequences of all 38 larvae (18 females, 20 males) of S. bergi were analyzed completely. The general features of the polytene complement conformed to the photograph by
Chromosome arm IS of Simulium bergi (male larva), representing the Simulium subgeneric standard. Limits of two autosomal inversions and a puff are indicated with brackets on the standard sequence. CI = centromere of chromosome I.
Composite map of chromosome arm IIL of Simulium bergi (female larva), representing the Simulium subgeneric standard. Breakpoints of sex-linked inversions are indicated with brackets or arrows. Ordering the two independent sets of chromosome fragments indicated by the letters “a” through “h” will produce the inverted sequence for IIL-39,40 and IIL-49,50. CII = centromere of chromosome II, hc = insertion point for heterochromatic block, Pb = parabalbiani, 2ºNO = location of secondary nucleolar organizer, * = insertion point for 7 additional bands (only when IIL-41 is present).
Distal half of chromosome IIIL of Simulium bergi (female larva), representing the Simulium subgeneric standard. Limits of autosomal inversion IIIL-10 are indicated with a bracket on the standard sequence; cs = cup and saucer marker.
Fixed inversions. The fundamental banding sequence common to all larvae was derived from the standard subgeneric sequence by a single fixed inversion, IIS-C (sensu
Autosomal polymorphisms. Six autosomal polymorphisms were discovered (Table
Frequency of homologues with autosomal rearrangements in two populations of Simulium bergi.
ARMENIA | TURKEY | |
---|---|---|
Larvae (n) | 8 | 30 |
Homologues (n) | 16 | 60 |
IS-17† | 0.00 | 0.02 |
IS-18 | 0.06 | 0.00 |
IS-puff(13) | 0.06 | 0.00 |
IL-hb26 | 0.06 | 0.00 |
IL-hb(telomere) | 0.00 | 0.02 |
IIIL-10 | 0.44 | 0.08 |
Sex chromosomes. IIL was inferred as the sex arm in the Turkish population (Table
Chromosome arm IIL of male larvae of Simulium bergi. A Distal half of chromosome, showing heterozygous expression of the common Y-linked inversion IIL-22 B Complex sex-chromosome configuration, showing one homologue with IIL-39,40 and the other with IIL-41,52+hc71+7 extra bands; hc = heterochromatic block, Pb = parabalbiani, * = 7 additional bands inserted in one homologue.
Distribution of breakpoints of 36 sex-linked inversions in the IIL arm of Simulium bergi. Breakpoints are plotted according to section number of the standard banding sequence for the subgenus Simulium. Breakpoints falling at the junction of two sections are tallied for the distalmost of the two sections.
Number of larvae with each sex-linked rearrangement in two populations of Simulium bergi.
Armenia | Turkey | X or Y Linked† | |
---|---|---|---|
Females: Males | 5:3 | 13:17 | – |
IIL-21‡ | 1 | ||
IIL-22 | 1 | 13 | Y |
IIL-23 | 1 | 3 | X, Y§ |
IIL-24 | 2 | ||
IIL-25 | 2 | X | |
IIL-26 | 2 | X | |
IIL-27 | 1 | ||
IIL-28 | 3 | ||
IIL-29 | 2 | X | |
IIL-30 | 4 | X | |
IIL-31 | 2 | ||
IIL-32 | 1 | ||
IIL-33 | 1 | X | |
IIL-34 | 2 | Y | |
IIL-35 | 1 | X | |
IIL-36 | 1 | X | |
IIL-37 | 1 | X | |
IIL-38 | 1 | ||
IIL-39 | 2 | ||
IIL-40 | 1 | ||
IIL-41 | 1 | ||
IIL-42 | 1 | X | |
IIL-43 | 3 | X, Y | |
IIL-44 | 3 | 1 | X |
IIL-45 | 1 | ||
IIL-46 | 1 | ||
IIL-47 | 1 | ||
IIL-48 | 1 | ||
IIL-49 | 1 | X | |
IIL-50 | 1 | X | |
IIL hc71 | 1 | ||
IIL extra bands| | 1 | ||
IIL-51 | 1 | X | |
IIL-52 | 1 | Y | |
IIL-53 | 1 | X | |
IIL-54 | 1 | X | |
IIL-55 | 1 | X | |
IIL-56 | 2 | Y | |
IIL hc70 | 1 | X | |
IIL hc70/71 | 1 | X | |
IIL 2ºNO | 1 | X |
Chromosomal insights into taxonomy. We consider our Armenian and Turkish populations of S. bergi conspecific, based on shared chromosomal characters, viz., the entire fixed banding sequence, autosomal polymorphism IIIL-10, and four sex-linked inversions. Rearrangements unique to Armenia or Turkey probably reflect, in large part, small sample sizes. We would not expect restricted gene flow, given the flight capabilities of simuliids (
The presence of IIS-C chromosomally confirms the original (
Chromosomal fragility in the sex arm. Simulium bergi represents the most extreme known case of sex-chromosome differentiation in the Simuliidae, with 41 sex-linked rearrangements discovered on IIL (i.e., the sex arm) among 38 larvae. IIL distal to section 58, thus, is an area of rearrangement hotspots, particularly in the central region of the arm. This area of fragility includes sets of mimic inversions, two or more sequence reversals that resemble one another, differing by as little as one band (
Widespread species can exhibit a significant cumulative degree of sex-chromosome polymorphism over their entire geographical distribution, with inversions often replacing one another across the distribution (e.g., S. vittatum, Zetterstedt 1838;
In species with multiple rearrangements linked to sex, the sex-differential region tends to become progressively enlarged (
At least two inversions, IIL-23 and IIL-43, were linked predominantly to the X, but in one larva each, they were linked to the Y. Sex exceptions in the Simuliidae are frequent (e.g., Rothfels & Featherston 1981). They have been considered ancestral relicts, the result of crossing over, or products of transposable element excision (
The concentration of inversions in particular areas of the macrogenome, such as the IIL arm of S. bergi, emphasizes that inversions are not random. Inversion concentrations have been known and easily visualized for decades in the polytene chromosomes of dipterans (e.g.,
We draw attention not only to clustered inversions in the distal three-quarters of the IIL sex arm, but also to the clustering of other rearrangements. Five structural phenomena, other than inversions, such as heterochromatic blocks, are found in the sex arm (15% of the polytene complement) compared with three in the autosomal portion (85% of the complement). If the pattern is consistent, it suggests that models accounting for increased inversion frequency also should accommodate the increased frequency of nonbreak-type rearrangements, such as the addition of heterochromatin. The functions of heterochromatin are varied and include suppression of recombination (
What is not known is whether a particular area of the complement—the IIL arm of S. bergi, for example—is more susceptible to breakage or if the breaks that occur are more likely to persist through subsequent generations, or if both phenomena play a role. Also unknown is whether a visualized break shared by two or more inversions is equivalent at the level of the base pairs. This question can be addressed through molecular characterization of the distal and proximal breakpoint sequences (
We thank J. Martin, P. Michailova, W.S. Procunier, and an anonymous reviewer for helpful comments on the manuscript. This work was funded, in part, by National Science Foundation award DEB-0933218 (MIDGEPEET: A Collaborative Effort to Increase Taxonomic Expertise in Understudied Families of Nematocerous Diptera), and the Erciyes University Scientific and Technological Research Center Project TSA-2015-6011. Support for collecting the sample in Armenia was provided by the Civilian Research and Development Foundation to PHA and the late E. A. Kachvoryan. This is Technical Contribution No. 6418 of the Clemson University Experiment Station, based in part on work supported by NIFA/USDA under project number SC-1700433.