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 (White 1978, Rothfels 1989, Nevo 2012). If the chromosomes have played an integral role in the speciation process, individual species might be expected to carry unique signatures in their karyotype. In the dipteran family Simuliidae, the vast majority of species are chromosomally distinct from one another, even when they cannot be distinguished reliably by morphological criteria (Adler et al. 2010). Detailed banding sequences of the polytene chromosomes in the larval salivary glands of Simuliidae, consequently, have facilitated the discovery of cryptic species, provided insights into population structure and evolutionary relationships, and positioned the Simuliidae at the forefront of knowledge about the genetics of natural populations of insects (Adler and Crosskey 2015a).

The Prosimulium hirtipes group is a widespread Holarctic clade of the Simuliidae, consisting of 25 species in the Palearctic Region (Adler and Crosskey 2015b, Adler and Şirin 2015). Twelve of these species have been examined chromosomally, though to various degrees of precision (reviewed by Adler and Crosskey 2015a). Four species of the group occur in Armenia: Prosimulium frontatum Terteryan, 1956, Prosimulium petrosum Rubtsov, 1955, Prosimulium rachiliense Djafarov, 1954, and Prosimulium tomosvaryi (Enderlein, 1921) (Adler and Crosskey 2015b). Analyses of the polytene chromosomes of P. frontatum, P. rachiliense, and P. tomosvaryi have revealed cryptic biodiversity and provided hypotheses of their phylogenetic relationships (Adler and Şirin 2014). Comparative chromosomal studies of Armenian populations of P. petrosum, however, are lacking, although general karyotypic features, putatively of this species, have been presented for Bulgarian populations (Ralcheva and Dryanovska 1973, Ralcheva 1974, Chubareva and Petrova 2003).

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) (Rubtsov 1955). Adults attributed to this species were described from Azizbekov (= Vayk) in Armenia (Rubtsov 1956). Terteryan (1968), however, suggested that the descriptions of the Armenian adults represent P. pronevitshae Rubtsov, 1955, now a synonym of P. rachiliense (Crosskey and Zwick 2007, Adler and Şirin 2014). The pupal gill figured by Rubtsov (1956), based on Azerbaijanian material, has a branching formula of (2+2+2+2)+(2+2)+(2+2), whereas that by Terteryan (1968), based on Armenian material, has a formula of (3+3+2)+(2+2)+(2+2).

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 1). The material was fixed in a 3:1 mixture of ethanol and glacial acetic acid. Pupae and adults were not collected, but 13 mature larvae with well-developed gill histoblasts were obtained. Larvae were identified morphologically as P. petrosum, based on structural characters (Rubtsov 1956, Terteryan 1968)—the apex of the median hypostomal tooth of our material was posterior to the apices of the lateral teeth, and the 16 filaments of the pupal gill were arranged on three, widely splayed primary trunks, with a branching formula of (3+3+2)+(2+2)+(2+2) or (3+3+2)+(2+1+1)+(2+2).

Polytene chromosomes from the larval salivary glands were stained using the lacto-aceto orcein method (Bedo 1975), which also stained gonadal tissue. Preparations were spread by squashing chromosomes on a microscope slide. Larval gender was determined by the form of the gonads: rounded in males and elongated in females. Representative chromosomal arms and selected rearrangements were photographed under oil immersion (Figs 1–4). Composite digital images from different focal planes were made with Helicon Focus 5.3 and further processed with Adobe Photoshop CS6. The banding sequences of all six chromosomal arms were compared with the standard maps of the P. hirtipes group (Basrur 1962) and with maps of various species in the P. hirtipes group (Basrur 1959, Adler and Belqat 2001, Adler and Şirin 2014).

Figure 1.

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.

Figure 2.

Chromosome I of Prosimulium petrosum, with male and female transformed centromere regions (CI_t). Breakpoints of autosomal heterozygous inversions are indicated by brackets. C: centromere, NO: nucleolar organizer, 20/21hc: heterochromatic band.

Figure 3.

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.

Figure 4.

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 Basrur (1959). Newly discovered inversions were numbered to follow the last number assigned to inversions in other species of the P. hirtipes group currently under study and as yet unpublished. Chromosomal terminology, including terms for landmarks, follows that of Basrur (1959, 1962).

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’ (sensuAdler and Şirin 2014). The other 63 larvae (16 males, 47 females) were assigned to P. petrosum. All larvae had a diploid number of 2n = 6, with tightly paired homologues (Fig. 1).

The chromosomes were submetacentric (Fig. 1). Chromosome I (sections 1–44) was the longest, with the two arms (IS and IL) subequal in length, followed by chromosome II (sections 45–74) with the long arm (IIL) slightly longer than the short arm (IIS). Chromosome III (sections 75–100) was the shortest, with the long arm (IIIL) approximately 35% longer than the short arm (IIIS). The centromere regions of chromosomes I and II were transformed (CI_t, CII_t; sensuBasrur 1959), producing an expanded, flocculent area from section 19 through section 21 (CI_t) and from the middle of section 57 through section 58 (CII_t) (Figs 1–3). The centromere region of chromosome III was not expanded (Figs 1, 4).

A single, primary nucleolus organizer was in the standard position for the P. hirtipes group, that is, in the base of IL (Fig. 2). Landmarks that remained in the standard positions for the P. hirtipes group included the single Balbiani ring in the base of IIS (Fig. 3), “blister” in IIIS (Fig. 4), and “shield” and “triad” in IIIL (Fig. 4). A chromocenter was lacking, and supernumerary (B) chromosomes were absent.

Fixed (interspecific) inversions. The banding sequence of chromosome arms IS, IL, IIIS, and IIIL was identical with the standard banding sequence established by Basrur (1959, 1962) for the P. hirtipes group. Chromosome II, however, had four fixed inversions relative to the standard sequence—two overlapping inversions in the short arm, IIS-6 and IIS-7, and two tandem inversions in the long arm, IIL-9 and IIL-10 (Fig. 3). The four inversions involved 73% and 58% of the sections of IIS and IIL, respectively. IIL-9 moved the “group of 5” marker more centrally and IIL-10 reversed the polarity of the parabalbiani.

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 2, Figs 2, 3, 4). Four inversions in sections 96–100 of IIIL formed a complex set of loops collectively referred to as IIIL-dif (Fig. 4, e). The proposed breakpoints of these four inversions are shown in Fig. 4, f. All autosomal polymorphisms were expressed in low frequency (< 0.065; Table 2).

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 CI_t region, typically from section 20 through the beginning of section 21 (Fig. 2), although one male was unpaired from the beginning of section 19 to the beginning of section 21; no inversion could be discerned in the unpaired region. Females lacked the heterochromatic band and exhibited complete pairing of homologues in the CI_t region. Thus, the expanded centromere region of chromosome I was the sex-differential segment, with males X₀Y₁ and females X₀X₀. In two males, ectopic pairing of CI_t and CII_t occurred in some nuclei.

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 (Terteryan 1968), rather than to Azerbaijanian material (Rubtsov 1956), of P. petrosum. Based on hypostomal structure, they precisely match the Azerbaijanian (holotype) (Rubtsov 1956). Our Armenian collections and the type locality of P. petrosum in Azerbaijan are 140–160 km apart, and all are in the same ecoregion—the Caucasus Mixed Forests Ecoregion (World Wildlife Federation 2015). Although a slight difference in the branching pattern of the gill between Armenian and Azerbaijanian samples possibly indicates the presence of cryptic species, we attribute the difference to intraspecific variation, which is common, especially on the dorsal trunk, in members of the P. hirtipes group (Stloukalova 2004). Similarly, although we found mature larvae 1.0–1.5 months earlier (end of April–beginning of May) than did Terteryan (1968), the seasonal difference could be attributable to altitude or perhaps climatic variation among years. Given the minimal geographic distance, dispersal ability of simuliids (Adler et al. 2005), identical ecoregion, and morphological similarity, we conclude that our Armenian populations are conspecific with the holotype.

Although our material corresponds with the type (Caucasican) concept of P. petrosum (Rubtsov 1955), a larger question is whether P. petrosum is a unique species or conspecific with P. latimucro, as suggested by Adler and Crosskey (2015b), based on morphological similarity. Yankovsky (2003) suggested that the projection of the median hypostomal tooth anterior to the lateral teeth and the second-order branching (i.e., 3+3+2) of the upper gill trunk distinguish the larva of P. latimucro from that of P. petrosum. Accordingly, our samples correspond with P. petrosum, based on the hypostomal teeth, and with P. latimucro, based on the branching of the gill.

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 Basrur (1959), and fixation of IIL-10 with P. rufipes, P. “aff. 3”, and Moroccan P. latimucro (Adler and Belqat 2001, Adler and Şirin 2014). In European populations of P. latimucro, inversion IIL-10 is absent or polymorphic. Prosimulium petrosum differs from P. rufipes, P. “aff. 3”, and Moroccan P. latimucro by lacking IS-18, IIS-8, and IIL-11, respectively. Prosimulium petrosum does not share any autosomal polymorphisms with any studied member of the P. hirtipes group. We conclude that European populations of P. latimucro are most closely related to P. petrosum.

Males and females of P. petrosum consistently differ in the expression of their CI_t 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) (Rothfels 1980, Post 1982). Any of the three chromosomes (I, II, or III) can function as the sex chromosome. Identical, differentiated sex chromosomes are rarely shared between species (Rothfels and Freeman 1983, Adler et al. 2015). Thus, the sex chromosomes can be useful in species discovery and identification (Rothfels 1989).

Lack of pairing of homologues in the CI_t 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 (Basrur 1959, Adler unpublished). The heterochromatic band 20/21hc of P. petrosum also appears on the Y chromosome of various members of the P. hirtipes group, including Moroccan populations of P. rufipes and P. latimucro and some European populations of P. latimucro and P. “aff. 3”, often with various repatternings of banding in the CI_t region (Adler and Belqat 2001, Adler unpublished). No conspicuous repatterning was observed in the CI_t region of P. petrosum. We do not know if the unpaired CI_t condition and heterochromatic band 20/21hc are identical across populations and species, and if so, if their shared nature reflects common ancestry, introgression, or independent origins. Species differences in other members of the P. hirtipes group, such as those in eastern North America often are based on minor differences in the centromere region, especially of CIII (Rothfels and Freeman 1977).

A Y chromosome based on an unpaired CI_t 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 Ralcheva (1974) of putative P. petrosum from Bulgaria suggests that IS and IL are standard, IIS-6,7 and IIL-9 are present, and IIL-10 is absent; the sex chromosomes and larval morphology were not mentioned. Based on available evidence, P. petrosum of Ralcheva (1974), therefore, is probably P. latimucro, and most closely resembles populations in the Swiss Alps (as P. inflatum “aff. 1” of Basrur 1959). The chromosomal characteristics of all other analyzed populations identified as P. petrosum and P. latimucro are entirely congruent with the respective configurations of the hypostomal teeth. Thus, we argue that P. petrosum is a distinct species on the basis of unique chromosomal features corroborated by distinct hypostomal features. Prosimulium petrosum and European P. latimucro, therefore, are homosequential species—they have the same fixed banding sequence but differ morphologically, a phenomenon first discovered in Drosophila (Carson et al. 1967). Although not common in the Simuliidae, previous examples of homosequential species include several members of the Simulium vernum group (Hunter 1987, Adler et al. 2004, Seitz and Adler 2015).

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.