Black flies (Diptera: Simuliidae) are important to science because, in many cases, the single morphospecies of classical taxonomy reveals itself as any number of cytologically differentiable sibling species when larval polytene chromosomes are analyzed (Rothfels 1956). In fact, the presence of reproductively isolated sibling species among presumed single morphospecies of simuliids, and their subsequent taxonomic description as valid biological species, would have gone unrecognized had it not been for the initial cytogenetic analyses (Rothfels 1979). Generally, sex-linked paracentric inversions in males initially characterize cytologically differentiating taxa (Rothfels 1979). This pattern has been observed in numerous species complexes of black flies including Prosimulium hirtipes (Rothfels 1956), Simulium pictipes (Bedo 1975), S. venustum/S. verecundum (Rothfels et al. 1978), S. vittatum (Rothfels and Featherston 1981), S. arcticum (Shields and Procunier 1982), Prosimulium (Helodon) onychodactylus, (Newman 1983), Eusimulium pugetense (Allison and Shields 1989), E. aureum (Leonhardt and Feraday 1989), and S. tuberosum (McCreadie et al. 1995).

TABLE 1.

Taxa of the Simulium arcticum complex.

Nine sibling species and 16 cytotypes have been described within the S. arcticum complex (Shields and Procunier 1982, Procunier 1984, Adler et al. 2004, Shields et al. 2007a, 2007b, Conflitti et al. 2010; Table 1). The present study summarizes detailed cytogenetic analyses of nearly 15,000 larvae of the Simulium arcticum complex taken from 234 collections at 58 sites in Montana (Fig. 1), northern Idaho, Washington State, and Oregon (Fig. 2). I report frequencies of 20 taxa of the Simulium arcticum complex, distributions of these taxa, linkage of chromosomal inversions to X and Y chromosomes, and very rare chromosomal variation, including frequencies of pericentric inversions, chromosomal translocations, and numbers of polyploids. The present study approximates in scope the extensive studies of S. vittatum (Rothfels and Dunbar 1953, Rothfels and Featherston 1981) and of S. damnosum (Dunbar 1966, 1969, Vajime and Dunbar 1975). This study supports original observations by Rothfels (1989) which suggested that a continuum from cytotypes to cytospecies (taxa that have unique Y chromosomes and are reproductively isolated from each other) to morphospecies (taxa that have unique Y chromosomes and that can be identified on morphological grounds) may be occurring in black flies. Some of the data included in this broad comparative study have been reported in studies of correlates of genetic and environmental variation (Shields et al. 2007a) and assessments of the reproductive status of taxa in sympatry (Shields et al. 2007b, 2009, Shields and Kratochvil 2011). In the present study, I hypothesized that siblings would occur in high frequencies and have broader geographic distributions than cytotypes. I also hypothesized that linkage to the Y chromosome of various paracentric inversions in siblings and cytotypes would essentially be complete.

Fig. 1.

Locations of collections made in the state of Montana. The size of the circles corresponds to the number of collections made at that site (see collection key). The number in each circle corresponds to the site number listed in Appendix 2.

Fig. 2.

Locations of collections made in Idaho, Washington, and Oregon. The size of the circles corresponds to the number of collections made at that site (see collection key). The number in each circle corresponds to the site number listed in Appendix 2.

TABLE 2.

Frequencies of the 20 taxa of the Simulium arcticum complex observed in this study. Siblings are bolded.

METHODS

Methods of larval collection, fixation, morphological identification, staining of polytene chromosomes and gonads, and chromosome analyses have been presented elsewhere (Rothfels and Dunbar 1953, Shields and Procunier 1982, Currie 1986, Adler et al. 2004). I recorded the following for each sibling species and cytotype: collection locations (58 freshwater sites), dates of collection, water temperatures, GPS coordinates, and numbers of each taxon collected at that site and date (Appendixes 1, 2). Although some sites were re-collected for various reasons (e.g., Clearwater River in 2007, 2008 and 2009), such as for determinations of annual continuity of taxa (Shields et al. 2009), we generally sampled all streams and drainages in the larger study area. I used contingency analysis and the chi-square (χ²) test for geographic associations between taxa. Sizes of the geographic ranges were calculated using the minimum convex polygon method and ArcGIS 10.0 software. Although contingency tests assume nearly equal sample sizes, it is essentially impossible to obtain equal sample sizes of larvae. The number of larvae at any site at any time is influenced by many biological and environmental factors beyond our control. Taxa having fewer than 10 individuals were excluded from these analyses since small sample sizes could erroneously affect conclusions.

RESULTS

Linkage of Taxon-Specific Paracentric Inversions to the Y Chromosome

I analyzed 7483 male larvae of the S. arcticum complex, which included 17 taxa (Table 3). Ten of these taxa had complete linkage of their taxon-specific inversions to the Y chromosome, while 6 of the 7 remaining taxa had very high linkage to the Y (range of proportion of linkage 0.967–0.997). One female was a IIL-13 homozygote, 8 females were IIL-13 heterozygotes, and 22 males were IIL-13 heterozygotes. More study is needed to determine if this inversion is sex-linked.

Fig. 3.

Paracentric inversion breakpoints associated with the new cytotypes discovered on the IIL chromosome in this study. Larger numbers delineate sections of the long arm of chromosome II. The IIL-73•74 cytotype has 2 separate paracentric inversions associated with its Y chromosome. Inversions IIL-51, IIL-68, and IIL-73•74 were described as cytotypes in Conflitti et al. (2010), but limits of these inversions were not indicated in that publication.

Fig. 4.

The IIS chromosome arm. Larger numbers delineate sections of the short arm of chromosome II. The taxon specific IIS-12 inversion is shown in brackets.

TABLE 3.

Linkage to the Y chromosome of taxon-specific inversions in the Similium arcticum complex.

TABLE 4.

Distribution of the IIL-2 inversion among females and males of this study.

Simulium saxosum and Simulium apricarium

Females of Simulium saxosum tended to be IIL-2 inversion homozygotes, while males tended to be IIL-2 heterozygotes (Shields and Procunier 1982, Adler et al. 2004). Slightly more than 5.0% of S. saxosum females were heterozygotes for the IIL-2 inversion (Table 4). Simulium apricarium is characterized by the IIL-7 inversion and fixation of the IIS-11 autosomal inversion. IIL-7 occurs in all classes (st/st, st/i, and i/i) of both sexes (Adler et al. 2004, Shields et al. 2007a, 2007b). I analyzed more than 1600 S. apricarium in this study and found a significant difference (P < 0.001) in the distribution of genotypes and sex (Table 5). Far fewer st/i and i/i females and far fewer st/st and i/i males than expected were observed (Table 5).

TABLE 5.

Distribution of the IIL-7 inversion among female and male Similium apricarium (G = 60.63262, df = 2, P ≤ 0.001).

TABLE 6.

Incidence of B chromosomes in taxa of the Similium arcticum complex.

DISCUSSION

I hypothesized that when a large geographic sample was obtained, previously described siblings (Shields and Procunier 1982, Adler et al. 2004) would occur in higher frequencies and have larger geographic distributions than cytotypes, and the findings presented here support that hypothesis. The 5 siblings observed here accounted for 69% of all males analyzed. These observations correspond to those of Adler et al. (2004:814–822), who observed large geographic distributions for 8 of 9 cytospecies of the S. arcticum complex. In my study, the sibling S. negativum IL-3•4 had an intermediate frequency of males and a small geographic distribution, but these findings may relate to the sibling's later emergence in summer in our study area when we tended to collect larvae less frequently. Even though there were 15 cytotypes described here, they accounted for less than a third of males. Correspondingly, all of the 15 cytotypes observed had intermediate to very small geographic distributions, and of these, 6 were found at only one site. Though the reproductive statuses of all taxa of the S. arcticum complex have not been determined, 4 independent studies (Shields et al. 2007a, 2007b, 2009) showed that siblings in sympatry are reproductively isolated while cytotypes in sympatry are not. I thus conclude that (1) siblings may be more abundant and may have larger geographic distributions than do cytotypes and (2) siblings in sympatry are reproductively isolated while cytotypes are not.

TABLE 7.

Tests of association between taxa regarding geographic distribution. Bold type indicates highly significant positive geographic associations; italic type indicates highly significant negative geographic associations.

TABLE 8.

Frequencies of very rare sex-linked paracentric inversions within the Similium arcticum complex. This analysis does not include populations of Simulium saxosum and S. apricarium since those taxa have different distributions of X and Y linkage.

TABLE 9.

Frequencies of pericentric inversions, translocations, and polyploids within the Similium arcticum complex.

I also hypothesized that taxon-specific inversions within the S. arcticum complex would be completely linked to chromosomes determining sex. Most of the types observed here had extremely tight linkage to their respective Y chromosomes. Ten of the types have complete linkage to Y, while the remaining 6 have an average linkage of 0.985. These findings strongly suggest that chromosomal inversions related to sex determination in the S. arcticum complex possibly play an early and significant role in the speciation process. Though Coyne and Orr (2004:265) may be correct when they state, “It is far from clear if chromosomal speciation is common in animals generally; indeed, we know of no compelling evidence for chromosomal speciation in animals,” this may not be the case in black flies. As stated earlier, Y-linked inversions are common in complexes of black flies, and based on our assessments of molecular divergence within the S. arcticum complex (Conflitti et al. 2010, 2012), these inversions may occur early in the speciation process. Thus, based on our cytogenetic analysis of the largest sample ever reported for a complex of black flies, we expand on a model for chromosome-based speciation in black flies that was initially proposed by Rothfels (1989; Fig. 5). If this chromosome-based model is relevant, we should possibly observe a continuum of taxa whose earliest members (cytotypes) can be distinguished only by unique inversion linkage to the Y chromosome, and whose later members are either intermediate in the speciation process or are distinctive morphospecies.

As mentioned earlier, S. apricarium and S. saxosum are exceptions to the “male Y-linkage” paradigm. The taxon-specific IIL-7 inversion in S. apricarium occurs in all genotypic categories (st/st, st/i, and i/i) of both females and males. The tendency for both females and males to possess IIL-7 heterozygotes as shown here may represent a novel and transitional form of sex-chromosome evolution. Sex in S. saxosum is determined by the inheritance of the X chromosome (the large majority of females being X_IIL-i X_IIL-i, while all males are X_IIL-i Y_IIL-st). As with the situation in S. apricarium, sex-linkage to the X in S. saxosum may represent a transitional state.

Fig. 5.

Chromosome-based model for speciation in the Simulium arcticum complex of black flies. The 15 cytotypes described here (Table 2) support step 1 of this model. Step 2 and the designation of assortative mating in the model are supported by the presence of a remnant population of S. arcticum s. s. and S. saxosum at the Coeur d'Alene River, Idaho (Shields and Kratochvil 2011). Step 3 is supported by fixation of the IIS-11 autosomal inversion among S. arcticum s. s. and S. apricarium at Little Prickly Fear Creek (Shields et al. 2007b). Step 4 is supported by temporal reproductive isolation of S. negativum and S. arcticum IIL-9 at the Blackfoot River and by sympatric reproductive isolation of S. arcticum s. s. and S. apricarium at Little Prickly Fear Creek (Shields et al. 2007b) Designations of DNA non-monophyly and monophyly are based on Conflitti et al. (2010, 2012).

I am able to morphologically separate larvae of Simulium negativum from other members of the S. arcticum complex based on the negative head patterns of females (Adler et al. 2004) and the overall light and fragile appearance of larvae. Correspondingly, unlike all other members of the Simulium arcticum complex, sex in S. negativum is based on inversions in the long arm of chromosome I (Shields and Procunier 1982, Adler et al. 2004). This difference indicates that Simulium negativum may (1) be the most divergent member of the complex, (2) be a validly described morphospecies, and (3) be near the temporal apex of a chromosome-based speciation model (Fig. 5; see below regarding DNA evidence).

Our earlier studies support such a contention. We observed that (1) 2 siblings, S. arcticum s. s. and S. apricarium, were reproductively isolated in sympatry; (2) a sibling, S. negativum, and a cytotype, S. arcticum IIL-9, were temporally reproductively isolated; and (3) 2 cytotypes, S. arcticum IIL-9 and IIL-19, were not reproductively isolated in sympatry (Shields et al., 2007b). Moreover, a sibling, S. arcticum s. s., and a cytotype, S. arcticum IIL-22, were not reproductively isolated in sympatry (Shields et al. 2009). Such combinations of reproductive status support a continuum model of chromosomal speciation.

Rothfels (1989) proposed that speciating taxa having unique Y-linked chromosomal inversions within a black fly complex might undergo initial mating trials. These mating trials eventually lead to coadaptation of polymorphic sex chromosomes in pairs, followed by reinforcement through assortative mating and slight selective advantage. In such a case, we might expect to find geographically parapatric taxa that still share combinations of sex chromosomes in areas of overlap. Indeed, we have found such a case in which the westerly distributed S. saxosum and the easterly distributed S. arcticum s. s. still share combinational sex chromosomes, interpreted as the “remnants of mating trials,” in a small area of overlap at the Coeur d'Alene River in northern Idaho (Shields and Kratochvil 2011).

We know that the majority of taxa within the S. arcticum complex appear relatively young in an evolutionary sense because they are not monophyletic in DNA sequence trees based on comparisons of mitochondrial and nuclear genes (Conflitti et al. 2010, 2012). Correspondingly, we might expect to observe morphologically and chromosomally distinct species at the far end of the speciation continuum model. As stated above, S. negativum is morphologically distinct (negative head patterns in females; Adler et al. 2004) and chromosomally distinct; males are IL-3•4 heterozygotes (Shields and Procunier 1982). Correspondingly, S. negativum is molecularly monophyletic to the remainder of taxa of the S. arcticum complex (Conflitti et al. 2010, 2012). This attribute also corresponds to other biological features of S. negativum. As taxa of the S. arcticum complex are speciating through time, we might expect to observe environmental correlates with cytogenetic diversity. Indeed, the distributions of some taxa appear to be influenced by elevation, date of collection, and water temperature. My observations based on this large sample size support our earlier observation that S. arcticum IIL-18 occurs at high elevations and is possibly influenced by cooler water temperatures in spring (Shields et al. 2007a). However, our earlier observation (Shields et al. 2007a) that S. apricarium is restricted to low elevations and that both S. brevicercum and S. arcticum s. s. are distributed randomly with respect to elevation is not supported by this more extensive analysis. Adler et al. (2004) named the IIL-7 cytospecies S. apricarium, which literally means “of the open.” Simulium apricarium females possibly preferentially deposit their eggs in open (broader) streams rather than being influenced by elevation. Why female black flies oviposit at specific sites (Hunter and Jain 2000, Adler et al. 2004) deserves additional study.

Our data suggest that some taxa of the S. arcticum complex have highly significant positive associations with respect to geographic distribution, while other taxa do not. Both of the negative associations with respect to distribution involve only siblings, while 3 of the 4 positive associations involve both siblings and cytotypes. None of these taxa is monophyletic in DNA sequence trees (Conflitti et al. 2010, 2012), yet S. negativum is. More detailed study of S. negativum from both the cytogenetic and environmental perspectives appears warranted.

This study summarizes an analysis of a large sample of larvae of the S. arcticum complex across a broad geographic area. Inversions linked to sex chromosomes are omnipresent.

Frequencies and distributions of siblings are larger than those of cytotypes. Our previous studies indicate that siblings tend to be reproductively isolated when sympatric, while cytotypes are not. Our molecular studies indicate that the complex may be young in an evolutionary sense but that Simulium negativum is molecularly monophyletic with respect to other members of the complex. While my analysis is extensive, it covers roughly one twentieth of the distribution of the S. arcticum complex in western North America. Similar studies north and south of the northern Rocky Mountains are clearly needed.