Research Article |
Corresponding author: Sergey Vlasov ( sv.vlasov@mgou.ru ) Academic editor: Veronika Golygina
© 2016 Sergey Vlasov, Maria Harutyunova, Karine Harutyunova, Peter Adler.
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:
Vlasov S, Harutyunova M, Harutyunova K, Adler PH (2016) Chromosomal evidence of species status and evolutionary relationships of the black fly Prosimulium petrosum (Diptera, Simuliidae) in Armenia. Comparative Cytogenetics 10(1): 33-44. https://doi.org/10.3897/CompCytogen.v10i1.6551
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The karyotype of Armenian populations of the black fly Prosimulium petrosum Rubtsov, 1955 was characterized and compared with that of all other chromosomally known Palearctic members of the Prosimulium hirtipes group. Analysis of the polytene chromosomes established that Prosimulium petrosum is most closely related to European populations of Prosimulium latimucro (Enderlein, 1925) with which it shares an identical fixed chromosomal banding sequence. Its validity as a species, separate from Prosimulium latimucro, is based on its unique sex-differential sections in the expanded centromere region of chromosome I, in agreement with the unique structural configuration of the hypostomal teeth of its larvae. Prosimulium petrosum and Prosimulium latimucro, therefore, are homosequential species, demonstrating the value of a combined chromosomal and morphological approach in determining species status.
Black flies, chromosomal inversions, homosequential species, polytene chromosomes, sex chromosomes
Chromosomal rearrangements have long been considered a driving force in speciation in certain groups of organisms, based on a wealth of evidence, much of it indirect (
The Prosimulium hirtipes group is a widespread Holarctic clade of the Simuliidae, consisting of 25 species in the Palearctic Region (
Prosimulium petrosum was described from larvae and pupae collected on 26 May 1952 in Azerbaijan; the holotype larva is from River Agsu above Lake Göygöl (= Geigel) (
Given the lack of chromosomal information for bona fide material of P. petrosum, we conducted a comparative band-by-band analysis of P. petrosum to characterize its karyotype and illuminate its taxonomic status and evolutionary relationships. In particular, we were interested in determining if P. petrosum is a species distinct from the morphologically similar European species, P. latimucro (Enderlein, 1925), or if they are conspecific.
Larvae were collected from three streams, up to about 210 km apart, in April and May in northern and southern Armenia (Table
Site | Location | Latitude Longitude | Altitude (m asl) | Date | Larvae analyzed males:females |
---|---|---|---|---|---|
1 | Armenia, Gegarkunik Province, Ddmashen |
40°34'N 44°49'E | ca. 1900 | 21 April 2010 | 3:5 |
2 | Armenia, Sjunik Province, Mogralzani-Vardanidzor, Megraget River | 39°00.40'N 46°12.45'E | ca. 1265 | 04 May 2011 | 0:1 |
3 | Armenia, Sjunik Province, Megrinsky pass | 39°06.30'N 46°10.47'E | ca. 2375 | 04 May 2011 | 13:41 |
Polytene chromosomes from the larval salivary glands were stained using the lacto-aceto orcein method (
Total polytene chromosomal complement of Prosimulium petrosum. Total polytene chromosomal complement of female larva of Prosimulium petrosum, showing the diploid condition of 2n = 6, with tightly paired homologues.
Chromosome I of Prosimulium petrosum, with male and female transformed centromere regions (CIt). Breakpoints of autosomal heterozygous inversions are indicated by brackets. C: centromere, NO: nucleolar organizer, 20/21hc: heterochromatic band.
Chromosome II of Prosimulium petrosum. Relative to the standard sequence, fixed inversions IIS-6, IIS-7, IIL-9, and IIL-10 are present. Breakpoints of autosomal inversions are indicated by brackets above the chromosomes. BR: Balbiani ring, C: centromere, Pb: parabalbiani, “5”: group of 5 marker.
Chromosome III of Prosimulium petrosum. a, b chromosome III of Prosimulium petrosum. Breakpoints of autosomal inversions and location of 2 additional bands (87dp) are indicated by brackets. IIIL-dif is an inversion complex, hypothesized to consist of four inversions. Arrows indicate locations of 90hb and 100dlT c heterozygous band duplication 87dp d heteroband 90hb and heterozygous inversion IIIL-33 e complex set of heterozygous inversions, collectively referred to as IIIL-dif; arrow shows deletion 100dlT in telomere of one homologue. f - heterozygous deletion 100dlT; breakpoints of individual inversions IIIL-34, IIIL-35+IIIL-36+IIIL-37, which comprise complex inversion IIIL-dif, are indicated by brackets; dashed line designates approximate limits of inversions. C: centromere, bl: blister, ca: capsule, s: shield, tr: triad.
Fixed inversions (i.e., homozygous in all larvae) are italicized in the text and underlined on our maps; floating inversions (i.e., polymorphisms) are not italicized or underlined. Inversions identical to those identified in previous studies (i.e., IIS-6, IIS-7, IIL-9, and IIL-10) were given the same numbers assigned by
Three morphological preparations of mature larvae were deposited in the Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia. Additional morphological preparations and chromosomal photographs were deposited in the Moscow State Regional University, Moscow, Russia.
Karyotype. In total, 64 larvae were analyzed. One larva from Site 1 chromosomally matched the banding sequence of P. rachiliense cytoform ‘A’ (sensu
The chromosomes were submetacentric (Fig.
A single, primary nucleolus organizer was in the standard position for the P. hirtipes group, that is, in the base of IL (Fig.
Fixed (interspecific) inversions. The banding sequence of chromosome arms IS, IL, IIIS, and IIIL was identical with the standard banding sequence established by
Autosomal (intraspecific) polymorphisms. Fifteen autosomal polymorphisms were detected; all were present in the heterozygous state only. These autosomal rearrangements included 12 inversions, one heteroband (90hb), a duplication of bands (87dp), and a telomeric deletion (100dlT) (Table
Frequency of homologues with autosomal inversions and other rearrangements (band deletions, duplications, and heterobands) in three Armenian populations of Prosimulium petrosum.
Collection site | 1 | 2 | 3 | Armenia |
---|---|---|---|---|
Larvae (n) | 8 | 1 | 54 | 63 |
Chromosomal homologues (n)* | 16 | 2 | 108 | 126 |
IS-27 | 0.063 | 0.037 | 0.040 | |
IS-28 | 0.063 | 0.008 | ||
IL-16 | 0.063 | 0.009 | 0.016 | |
IIS-16 | 0.009 | 0.008 | ||
IIL-13 | 0.009 | 0.008 | ||
IIL-14 | 0.028 | 0.024 | ||
IIIL-32 | 0.019 | 0.016 | ||
IIIL-33 | 0.028 | 0.024 | ||
IIIL-dif |
0.028 | 0.024 | ||
100dlT |
0.046 | 0.040 | ||
90hb | 0.019 | 0.016 | ||
87dp | 0.009 | 0.008 | ||
Mean number of heterozygous inversions/larva |
0.333 | |||
Mean number of all heterozygous chromosomal rearrangements/larva |
0.460 |
Sex-differential region. All 16 males had a heterochromatic band (20/21hc) at the junction of sections 20 and 21 and lacked conjugation in the CIt region, typically from section 20 through the beginning of section 21 (Fig.
Our chromosomal analysis requires taxonomic context, especially a reasonable assignment of the correct species name. The larvae from our three sites in Armenia are chromosomally cohesive. Based on gill structure, they conform to previous Armenian collections (
Although our material corresponds with the type (Caucasican) concept of P. petrosum (
What do the banding sequences of the polytene chromosomes reveal about possible conspecificity of P. petrosum and P. latimucro and their evolutionary relationships? The Armenian population of P. petrosum shares IIS-6,7 and IIL-9 with P. latimucro, P. rufipes (Meigen, 1830), and P. “aff. 3” of
Males and females of P. petrosum consistently differ in the expression of their CIt region, indicating the general location of the sex-determining locus. The sex chromosomes of the Simuliidae often are associated with rearrangements, such as inversions and heterobands, although the X and Y also can be microscopically undifferentiated (X0Y0) (
Lack of pairing of homologues in the CIt region, observed in males of P. petrosum, also is found in at least some populations of other Palearctic members of the P. hirtipes group, such as P. hirtipes (Fries, 1824), P. latimucro, and P. “aff. 3”, and often serves as the basis for further elaboration of the Y chromosome, such as the addition of sex-linked inversions and heterobands (
A Y chromosome based on an unpaired CIt region, coupled with 20/21hc, without an associated inversion or band repatterning, uniquely characterizes P. petrosum. The allopatric nature of P. petrosum and P. latimucro, however, presents a challenge for evaluating reproductive isolation; the nearest chromosomally analyzed populations of P. petrosum and P. latimucro are more than 1,500 km apart. Our analysis of the photographs by
This work was supported by the International Scientific and Technical Center (ISTC) Project #A-1662 “Molecular Genetic Monitoring of Blood-Sucking Flies (Diptera) as a Basis for Biological Control of Vectors of Dangerous Infectious Diseases and Precautions against the Acts of Biological Terrorism”. We thank three reviewers for comments that improved the presentation of the paper.