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Incipient speciation and additional diversity within the Simulium arcticum complex of black flies (Diptera: Simuliidae).


In their seminal publication, Speciation, Jerry Coyne and Allen Orr (2004) suggested that it is important to investigate and understand processes that occur prior to and during a speciation event rather than to hypothesize potential mechanisms after the fact. They (2004; page 265) also suggested there is essentially no evidence that chromosome change in sympatric populations leads to speciation. However, Rothfels (1989) hypothesized the possibility of "mating trials" within sympatric populations of black flies (Simuliidae) between taxa differentiated by paracentric chromosomal inversions in males. Rothfels (1989) also predicted these mating trials would eventually give rise to cytologically distinct taxa and result in reproductively isolated biological species. Many morphospecies of black flies are in reality complexes of sibling species differentiable only when polytene chromosomes are analyzed (Shields and Procunier, 1982; Procunier, 1984; Rothfels, 1989; Adler et al., 2004). Shields and Procunier (1982) discovered five sibling species within the Simulium arcticum complex and four of these have been formally described as good biological species (Adler et al., 2004). Additionally, Shields (2013) has described 20 new cytotypes in the Pacific Northwest, groups having unique chromosomal inversions exclusively in males. We have tested Rothfels' chromosome model for speciation in black flies by comparing DNAs of taxa of S. arcticum in sympatry (Conflitti et al., 2010, 2011; Shields, 2013) and these observations suggest paracentric inversions in Y chromosomes of males occur early in the differentiation process and therefore may be important in the promotion of reproductive isolation.

Shields and Kratochvil (2011) discovered a population of black flies of the Simulium arcticum complex at the Coeur d'Alene River in northern Idaho that not only had sex chromosomes characteristic of S. arcticum s. s. and S. saxosum but also possessed combinations of the two. We referred to these types as "combinational" rather than "hybrids" since we were unsure of their origin (Shields and Kratochvil, 2011). Figure 1 describes the two alternative hypotheses for their origin (hybrid/secondary contact types as opposed to primary source/shared polymorphism types). The hybrid/secondary contact hypothesis implies that S. arcticum s. s. evolved in and to the east of the Rocky Mountains, while S. saxosum evolved separately in regions west of the Rockies. It also implies that combinational types described in this research are relatively recent and are the result of secondary contact between the aforementioned species. Alternatively, the primary source/ shared polymorphism hypothesis implies that combinational types are old and are the surviving ancestors (remnants) of mating trials between the two taxa.

Virtually all males of S. arcticum sensu stricto are heterozygotes for the IIL-3 inversion, [X.sub.0][Y.sub.IIL-3i] (third inversion found in the long arm of chromosome II), while females possess the standard, [X.sub.0][X.sub.0], noninverted sequence (Shields and Procunier, 1982; Shields, 2013). Alternatively, most males of S. saxosum are heterozygotes for the IIL-2 inversion, [X.sub.IIL-2i] [Y.sub.0], (second inversion found in chromosome II), while most females are IIL-2 inversion homozygotes, [X.sub.IIL-2i][X.sub.IIL-2i] (Shields and Procunier, 1982; Shields, 2013). The presence of: females heterozygous for IIL-2, of standard males, and of IIL-2/IIL-3 males at the Coeur d'Alene River suggested combinational sex chromosome types (Shields and Kratochvil, 2011). Moreover, the total absence of IIL-2 homozygotic males ([X.sub.IIL-2i][Y.sub.IIL.2i] and IIL-3 homozygotes of either sex ([X.sub.IIL-3i][X.sub.IIL-3i], [X.sub.IIL-3i] [Y.sub.IIL-3i]) indicated that these inversions were not acting autosomally (Shields and Kratochvil, 2011). There was no evidence that the combinational forms resulted from hybridization since the population at the Coeur d'Alene was in genetic equilibrium, did not exhibit loose pairing of homologues, nor were centromeres dimorphic (Shields and Kratochvil, 2011). We therefore tentatively described the population at the Coeur d'Alene River as a remnant of a past incipient speciation event and hypothesized that it eventually gave rise to S. saxosum to the west and to S. arcticum s. s. to the east (Shields and Kratochvil, 2011).

No other combinational populations were found among eight other populations to the east and west (Shields and Kratochvil, 2011). S. saxosum generally occurs in the western slopes of the Rocky Mountains while S. arcticum s. s. is generally found in the eastern slopes of the Rockies (Adler et al., 2004; Shields, 2013). Thus, the geographic zone of potential overlap between the two species runs primarily north and south. In the present study, I analyzed more than 1000 individuals of the S. arcticum complex from 37 collections to the south, northeast and northwest of the Coeur d'Alene River for sex chromosome constitution to further test whether types arose by recent hybridization or alternatively were remnants of a past incipient speciation event. New sites were analyzed to determine the geographic extent of this phenomenon. I also monitored the distribution of sex chromosome types to determine the equilibrium status of the larger populations of S. arcticum at the St. Joe River. Since we found only a single remnant population at the Coeur d' Alene River in our earlier study (Shields and Kratochvil, 2011), I hypothesized that we would not find remnants elsewhere.

The combination of our chromosome analyses (Shields and Kratochvil, 2011) and comparisons of DNAs of the various taxa of the S. arcticum complex (Conflitti et al., 2010, 2012) suggested that Y-linked inversions may occur early in the speciation process as Rothfels (1989) predicted. Molecular comparisons based on mitochondrial 12S, COII, cyt b and ITS-1 gene sequences (Conflitti et al., 2010) and on the mt COI, barcoding gene (Conflitti et al., 2012) resulted in nonmonophyly of chromosomally identified taxa of the S. arcticum complex, with the exception of S. negativum. These earlier observations provided the opportunity to investigate in greater detail a potential incipient speciation event within the S. arcticum complex.


I used conventional methods of polytene chromosome analysis (Shields and Procunier, 1982; Shields, 2013) to determine the sex chromosome constitution of larvae of the S. arcticum complex from 37 collections from 30 locations oriented generally in a north-south direction from the Coeur d'Alene River. Some collections were made in central Washington and northwestern Montana because these areas had not previously been sampled (Shields, 2013). At each site larvae were sampled from submersed rocks, branches, and twigs in the swiftest flowing water. I attempted to collect as broad an area as possible at each site to avoid collection of larvae from a single mated female. Larvae were immediately fixed in a 4 C solution of Carnoy's fixative which was changed thereafter until the supernatant became clear. In the laboratory larvae were sorted to species (Currie, 1986; Adler et al., 2004). Members of the S. arcticum complex were then opened to expose the salivary glands and gonads and stained in Feulgen (Rothfels and Dunbar, 1953). I used chromosome maps of the S. arcticum complex (Shields and Procunier, 1982) to determine sex chromosome variation and the genotypic frequency of the IS-1 autosomal polymorphisms (Shields et al., 2009). Microscope slides containing polytene chromosomes and gonads of each individual have been retained for future study, if necessary. For larger samples, such as those from the

St. Joe River whose chromosomes were of high quality: I analyzed all sex chromosome types in 2011, 2012, and 2013, using the Hardy-Weinberg equation, and the [X.sup.2] test to determine if populations each year were in genetic equilibrium. I used the minimum convex polygon method and Arc GIS 10.0 software to determine the size of the geographic area of combinational (putative remnant) types. The data are presented in three categories: (1) the non S. arcticum complex sites, (2) the S. arcticum complex combinational sites, and (3) the S. arcticum complex noncombinational sites.


Non S. arcticum complex sites.--Fourteen of the collection sites had either very small numbers or immature larvae of S. arcticum or those of other species (Table 1). They are not discussed further. Notably, 13 of the 30 sites possessed larvae of the Helodon onychodactylus complex (Adler et al, 2004).

S. arcticum complex combinational sites.--I found larvae of S. saxosum, S. arcticum s. s. and their combinational types in 11 collections (Table 2; Fig. 2). Larvae from Wolf Lodge Creek and the Fisher River though not abundant, possessed combinational types in all categories (Table 2). With the exception of the St. Joe River, 27.5 km. to the southeast and the Fisher River, 130 km. to the northeast most of these combinational sites were near the Coeur d'Alene River. I estimate that the geographic area of combinational sex-chromosome types is 3425 [km.sup.2].

Since S. arcticum larvae were abundant at the St. Joe River and since the quality of their polytene chromosomes was excellent, I compared chromosome diversity within and among samples taken in 2011, 2012 and 2013 (avg. n = 242; Table 3). Chromosomes that defined S. saxosum, S. arcticum s. s. and their combinational types were present in all three years (Table 3). Sex chromosome types abundant in one year were also abundant in the other years. Although the frequencies of the various sex chromosome types are significantly different from year-to-year ([X.sup.2] = 51.6, d. f. = 16, 0.0005 < P < 0.001), the populations are none-the-less, in genetic equilibrium (2011, [X.sup.2] = 12.1, d. f. = 8, P = 0.147; 2012, [X.sup.2] = 2.35, d. f. = 8, P = 0.968. The population at the St. Joe River in 2013 is: [X.sup.2] = 16.38, d. f. = 8, P = 0.037, however, the Bonferroni correction, alpha, is 0.05/3 = 0.017. Therefore, I conclude that the 2013 St. Joe population is in genetic equilibrium because the P value, 0.037 is larger than the Bobferroni significance level of 0.017. I observed no differences in pairing of homologues nor did I observe differences in the structure of centromeres (data not shown). Reduced pairing in combinational homologues and differences in the morphology of their centromeres could suggest hybridization.

A cytotype new to science, IIL-79, (males = [X.sub.0][Y.sub.IIL-79i], females = [X.sub.0][X.sub.0]) is not only abundantly present at the St. Joe each year (avg. 29.6% of males) but is also present in combination with IIL-2 and IIL-3 types each year (Table 3; Fig. 3). Cytotypic status is suggested for this type since all IIL-79 types were males (no [X.sub.0][Y.sub.IIL-79] females were observed). Although Ho for the IS-1 autosomal polymorphism was similar for IIL-2/IIL-3 types (Ho = 0.24) and IIL-79 types (Ho = 0.27) analyzed separately for all three years, the allelic frequency of IS-1 standard types (st/st) was 0.59 as opposed to 0.85 for the IIL-2/IIL-3 types. Thus, there may be a temporal component to the frequency of IS-1.

S. arcticum complex Noncombinational sites.--I analyzed more than 100 larvae from the Kootenai River in northwestern Montana (Table 4). The site is diverse, with larvae having six types of Y chromosomes, one of which (IIS-15) is new to science, fixed for the standard (st/st) genotype for the IS-1 autosomal polymorphism and is unique to the Kootenai River (Fig. 4). One male from the Kootenai was both IIS-15i and IIL-9i, suggesting mating trials between these two taxa.

Y chromosomes of larvae from Morrell Creek, and the Swan and Yaak rivers of northwestern Montana (Table 5) are similar to one another. In the aggregate, two types of X chromosomes and eight types of Y chromosomes are present. Though some of these types are in combination, none possessed combinational types for IIL-2/IIL-3. Larvae from Nason Creek in north central Washington State (Table 6) are S. saxosum. No combinational types were found there.


I observed sex-chromosomes of S. arcticum s. s., S. saxosum and their combination types at five geographic locations suggesting that the minimal geographic area in which potential remnant populations may occur is at least 3500 [km.sup.2]. I therefore reject my original hypothesis that combinational types would only exist at the Coeur d'Alene River and accept the fact that combinational types occur elsewhere.

Since larvae were abundant at the St. Joe River and since their chromosomes were of good quality the site was studied in detail. All species-specific classes of sex-chromosome types of S. arcticum s. s., S. saxosum and their combinational types occur at the St. Joe River in all three years. Moreover, there is no evidence that alternative forms of sex chromosomes for either species are functioning autosomally. That is, the absence of IIL-2 homozygotes in males and the absence of IIL-3 inversion homozygotes among 726 larvae negate this possibility and suggest remnant types. That combinational types were found at five sites in this study strengthens our contention that remnants of an incipient speciation event exist beyond the Coeur d'Alene River.

Unlike the population at the Coeur d'Alene River which was predominately S. arcticum s. s., S. saxosum and their combinational types (Shields and Kratochvil, 2011), the population at the St. Joe River contained 27% of male larvae that were S. arcticum IIL-79 ([X.sub.0][Y.sub.IIL-79]), a type new to science. IIL-79 Y chromosomes were not present among 15,000 larvae from 234 collections from 60 sites analyzed by Shields (2013) suggesting an origin for IIL-79 at or near the St. Joe. Also, at the St Joe site, 20 larvae were [X.sub.IIL.2] [Y.sub.IIL-79] and four were [X.sub.0] [Y.sub.IIL-3-IIL-79]. These combinational types suggest an association between S. arcticum IIL-79 and S. arcticum s. s. and between S. arcticum IIL-79 and S. saxosum and the possibility that mating trials between the three types have occurred.

Frequencies of the various sex chromosome types at the St. Joe River were significantly different each year. Possibly this can be explained by random sample error during the collection process. Also, [X.sup.2] tests can be strongly influenced by sample categories that have few individuals (e.g., [X.sub.IIL-2]/[X.sub.IIL-2]; [X.sub.IIL-2]/[Y.sub.IIL-79] and [X.sub.0]/[Y.sub.IIL-79]).

All three populations of S. arcticum at the St. Joe were in Hardy-Weinberg equilibrium for all categories of sex chromosome combinations suggesting that recent hybridization did not give rise to the combinational types there. Equilibrium also suggests that the presence of X arcticum IIL-79 types might not influence the random breeding of types at this site and that mating trials may have occurred. The decline in frequency of the standard IS-1 allele from 0.85 in IIL-2/3 types to 0.59 in IIL-79 types might alternatively suggest slight divergence between IIL-79 and the other two taxa.

My data are supported by previous cytogenetic work at taxon pure sites (e.g., S. saxosum, IIL-2, at the Cle Elum River and S. arcticum, IIL-3, at Little Prickly Pear Creek) in contrast to the combinational sites reported here (Table 7). The data are also supported by analyses of nuclear microsatellites which show panmixia of all taxa at the Coeur d'Alene site. In contrast, there are differences in microsatellite structure between S. saxosum at the Cle Elum River to the west and other populations of S. arcticum s. s. to the east when chromosome designations as priors are included in the microsatellite analysis (Conflitti et al., 2014, manuscript in review). I interpret these results to indicate that S. saxosum and S. arcticum s. s. may have arisen from a remnant population at the Coeur d'Alene River that may be ancestral to the two.

Inversions linked to the Y chromosome of black flies in general and specifically in the S. arcticum complex appear to be the first differentiating processes leading to reproductive isolation (Rothfels, 1989; Conflitti et al., 2010, 2012; Shields, 2013). Multiple chromosome inversions on the same Y chromosome are associated with maleness (e.g., IIL-2, IIL-79 and IIL-3, IIL-79) and indicate that the origin of novel rearrangements may eventually promote reproductive isolation.

Rothfels and Featherston (1981) observed overlap of the X vittatum sibling species, IS-7 and IIIL-1, across their range and suggested an origin for both in the Great Lakes region.

Hybridization and secondary contact were ruled out since these would require the reversal of two well differentiated sex chromosome systems and the remote possibility of the sharing of autosomal polymorphisms (Rothfels and Featherston, 1981). Contrary to Rothfels and Featherston (1981), Duncan et al (2004) used polymorphic DNAs and suggested IS-7 spread eastward, while IIIL-1 S. vittatum originated in the east and spread to the west. It is possible that the development of sex chromosome systems in black flies is dynamic and that types may arise or be lost.

Six types of male larvae of the S. arcticum complex occur at the Kootenai River in extreme northwestern Montana. The presence of numerous types of Fchromosomes at the Kootenai is similar to what is observed at other sites (Shields, 2013). In fact, diversity in sympatry rather than the existence of only one taxon/site appears to be the rule rather than the exception in the S. arcticum complex (Shields, 2013). Taxon-pure sites are rare within the complex, e.g., S. saxosum at the Cle Elum River (Shields, 2013). The Kootenai River and Rock Creek (Shields et al., 2007b) have IIL-9 and IIL-19 S. arcticum types in common which could be explained geographically since Rock Creek is only 230 km. to the southeast of the Kootenai.

The newly discovered IIS-15 S. arcticum constitutes the largest category of males at the Kootenai, yet it is found nowhere else (Shields, 2013) suggesting that new types may arise from other types of S. arcticum in sympatry. The presence of a IIS-15/IIL-9 male at the Kootenai suggests combinational types are present and that potential mating trials may be occurring between these taxa.

Types of sex chromosomes at Morrell Creek and the Swan and Yaak rivers are similar to those observed at the Clearwater River (Shields et al., 2009). Morrell Creek and the Swan River are 16.3 and 57.0 km. northwest of the Clearwater, respectively. However, the Yaak River is 264 km. northwest of the Clearwater with the dissimilar diversity of the Kootenai River in-between (Fig. 1). Data from Morrell Creek and the Swan and Yaak rivers are unique in that the IIL-22 and IIL-23 inversions occur solely in some individuals there. IIL-22 types occur at the Clearwater but IIL-23 types always occur with IIL-3 types there. Possibly these data suggest mating trials as well.

Data from Nason Creek in central Washington suggest the presence of S. saxosum ([X.sub.IIL-2i] [X.sub.IIL-2i] females and [X.sub.IIL-2i] [Y.sub.0] males). Most sites having members of the S. arcticum complex in western Washington are S. saxosum (Adler et al, 2004; Shields, 2013).

For taxa of S. arcticum described at the chromosome level, 22 have inversions associated with maleness in the long arm of chromosome II (Shields, 2013). Inversions linked to maleness also occur in the long arm of chromosome I (S. negativum, IL-3 x 4; Shields and Procunier, 1982; Adler et al., 2004) and in the short arm of chromosome II (S. arcticum ILS-4, Procunier, 1984; Adler et al, 2004; and S. vampirum, IIS-10 x 11, Adler et al., 2004, and IIS-15, as shown here). All IIS-15 larvae of this study were males and this suggests that short arm of chromosome II may also be a hot spot for inversions associated with sex in the S. arcticum complex.

Relevant to this study the major river drainages of central Idaho, south of the Coeur d'Alene and St. Joe rivers (e.g., the Clearwater, Lochsa, Salmon, Selway and Snake rivers) are unstudied with respect to the cytology of the S. arcticum complex and research on them will no doubt be informative to better understand the speciation process.

Detailed investigations, such as those of the present study, are revealing associations between sex-linked inversions, potential past mating trials and incipient speciation that has probably given rise to the currently allopatrically distributed and reproductively isolated species, S. arcticum s. s. and S. saxosum (Shields, 2013). Such studies aid in identification of ancestral and derived types in this diverse complex of black flies and provide a more detailed understanding of the diversification process.

Acknowledgments.--This research was supported by grants from the M. J. Murdock Charitable Trust (#2003198 and #2005233) and the National Geographic Society (NGS #7212-02) to Shields. Shields' research time was supported by the James J. Manion Endowed Chair of Biological Sciences Fund at Carroll College. I thank John and Pat Shields for help with collections and Dr. Grant Hokit of Carroll College for statistical advice. I thank Dr. Peter Adler, Clemson University, and an anonymous reviewer for commenting on an earlier draft of this manuscript.


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Department of Natural Sciences, Carroll College, 1601 North Benton Avenue, Helena, Montana 59625

(1) Corresponding author: Telephone: (406) 459-7644; FAX (406) 447-5476; e-mail:

TABLE 1.--Collection locations, dates and taxon information for
the black flies of this study

Location        GPS                      Date        Taxa present

1) Blue         48[degrees]34'25.5"N     4/28/2013   Helodon
Creek           115[degrees]35'06.0"W                onychodactylus
                                                     complex &

2) Cle          47[degrees]10'30.0"N     4/1/2013    No larvae
Elum River      121[degrees]01'40.0"W

3)              47[degrees]20'50.3"N     4/26/2013   H.
Clearwater      113[degrees]35T7.7"W                 onychodactylus
River,                                               complex
Mile 28
Highway 83

4) Coeur        47[degrees]34T0"N        3/25/2011   S. arcticum
d'Alene         116[degrees]16'25"W                  complex

Coeur           47[degrees]34'10"N       7/23/2011   S. arcticum
d'Alene         116[degrees]16'25"W                  complex

5) Edna         48[degrees]39'34.6"N     4/27/2013   No larvae
Creek           114[degrees]53'56.2,'W

6) Fernan       47[degrees]40T0"N        3/19/2011   Prosimulium
Creek           116[degrees]40T7"W                   pleurale

7)Fisher        48[degrees]21'46.0"N     4/27/2013   S. arcticum
River           115[degrees]19'13.2"W                complex

8) Fisher       48[degrees]04'10.3"N     4/28/2013   H.
River           115[degrees]22'26.6"W                onychodactylus
                                                     complex 8c S.

9) Flat         47[degrees]44'52"N       7/23/2011   S. arcticum
Creek           116[degrees]00'51"W                  complex

10) Foss        47[degrees]42'58.8"N     3/29/2013   H.
River           121[degrees]16'27.9"W                onychodactylus

11) Grave       48[degrees]47'27.3"N     4/27/2013   H.
Creek           114[degrees]55'59.1"W                onychodactylus

12) Icicle      47[degrees]33'04.4"N     3/29/2013   Immature
Creek           120[degrees]45'50.7"W                Prosimulium

13) Kootenai    48[degrees]22'03.4"N     4/27/2013   S. arcticum
River           115[degrees]19'24.5"W                complex

14) Kootenai    48[degrees]23'53.6"N     4/28/2013   S. arcticum
River           115[degrees]32'46.1"W                complex

15) Libby       48[degrees]13'32.1"N     4/28/2013   No larvae
Creek           155[degrees]28'39.8"W

16) Mile        47[degrees]52'26.4"N     4/26/2013   H.
67.5            113[degrees]50'02.5"W                onychodactylus
Highway 2                                            complex

17) Mile        48[degrees]0T55.9"N      4/28/2013   S. canonicolum
84.5            114[degrees]56.00.1"W
Highway 2

18) Morrell     47[degrees]08'45.4"N     4/26/2013   H.
Creek           113[degrees]27'56.3"W                onychodactylus
                                                     complex & S.

19) Nason       47[degrees]45'46.2"N     3/29/2013   5. saxosum
Creek           120[degrees]44'41.2"W

20) Pack        48[degrees]25'40"N       3/19/2011   P. formosum, H.
River           116[degrees]30'00"W                  onychodactylus
                                                     complex, 5.
                                                     canonicolum, S.
                                                     aureum complex
                                                     8c S. arcticum

21) St.         47[degrees]15'00"N       9/4/2006    S. arcticum
Joe R.          115[degrees]48'00"W                  complex

22) St.         47[degrees]16'25.6"N     3/18/2011   S. arcticum
Joe River       116[degrees]14'25.0"W                complex

St. Joe         47[degrees]16'25.6"N     3/26/2011   S. arcticum
River           116[degrees]14'25.0"W                complex

St. Joe         47[degrees]16'25.6"N     4/5/2012    S. arcticum
River           116[degrees]14'25.0"W                complex

St. Joe         47[degrees]16'25.6"N     4/8/2012    S. arcticum
River           116[degrees]14'25.0"W                complex

St. Joe         47[degrees]16'25.6"N     3/28/2013   S. arcticum
River           116[degrees]14'25.0"W                complex

23) South       48[degrees]47'18.9"N     4/28/2013   H.
Fork Yaak       115[degrees]39'57.7"W                onychodactylus
River                                                complex

24)             48[degrees]40'16.9"N     4/27/2013   H.
Stillwater      114[degrees]45'49.3"W                onychodactylus
River                                                complex

25) Swan        47[degrees]26'28.5"N     4/26/2013   S. arcticum
River           113[degrees]40'31.0"W                complex

Swan River      47[degrees]26'28.5"N     4/29/2013   S. arcticum
                113[degrees]40'31.0"W                complex

26) Tobacco     48[degrees]36'25.4"N     4/27/2013   H.
River           114[degrees]57'25.9"W                onychodactylus
                                                     complex & S.

27) Wenatchee   47[degrees]31T5.2"N      3/29/2013   S. arcticum
River           120[degrees]27'31.2"W                complex pupae

28) Wolf        47[degrees]37'00"N       3/19/2011   S. arcticum
Lodge Creek     116[degrees]36'20.0"W                complex

Wolf Lodge      47[degrees]37'00"N       3/25/2011   S. arcticum
Creek           116[degrees]36'20.0"W                complex

29) Yakima      47[degrees]18'28.4"N     4/1/2013    H.
River           121[degrees]18'53.9"W                onychodactylus
                                                     complex, S.
                                                     canonicolum, S.

30) Yaak        48[degrees]49'53.9"N     4/28/2013   H.
River           115[degrees]46T4.4"W                 onychodactylus

                                                     complex (sf S.

TABLE 2.--Sex chromosome diversity among combinational
sites of the S. arcticum complex


                              [x.sub.0]   [x.sub.0]   [X.sub.2]
Date                          [x.sub.0]   [x.sub.2]   [X.sub.2]

Coeur d' Alene R. 3/25/2011       1           0           0
Coeur d'Alene R. 7/23/2011        5           4           0
Wolf Lodge Cr. 3/19 & 3/
  25/2011                         6           1           1
Fisher R. 4/27/& 4/28/2013        4           4           0
Flat Cr. 7/23/2011                2           0           0
St. Joe R. 3/18 & 3/26,
  2011, 4/5 & 4/8, 2012
  & 3/28/2013                    217         63           2
St. Joe R. Avery 9/4/2006


                              [X.sub.0]   [X.sub.2]   [X.sub.0]
Date                          [Y.sub.0]   [Y.sub.0]   [Y.sub.3]

Coeur d' Alene R. 3/25/2011       0           1           4
Coeur d'Alene R. 7/23/2011        0           4           1
Wolf Lodge Cr. 3/19 & 3/
  25/2011                         2           3           3
Fisher R. 4/27/& 4/28/2013        5           1           3
Flat Cr. 7/23/2011                1           0           0
St. Joe R. 3/18 & 3/26,
  2011, 4/5 & 4/8, 2012
  & 3/28/2013                    52          54          160
St. Joe R. Avery 9/4/2006


                              [X.sub.0]   [X.sub.2]   [X.sub.2]
Date                          [Y.sub.9]   [Y.sub.3]   [Y.sub.9]

Coeur d' Alene R. 3/25/2011       0           2           0
Coeur d'Alene R. 7/23/2011       11           3           1
Wolf Lodge Cr. 3/19 & 3/
  25/2011                         0           2           0
Fisher R. 4/27/& 4/28/2013        0           1           0
Flat Cr. 7/23/2011               19           0           0
St. Joe R. 3/18 & 3/26,           0
  2011, 4/5 & 4/8, 2012
  & 3/28/2013                                46           0
St. Joe R. Avery 9/4/2006


                              [X.sub.0]    [X.sub.2]      [Y.sub.79]
Date                          [Y.sub.79]   [Y.sub.79]   [Y.sub.3 x 79]

Coeur d' Alene R. 3/25/2011       0            0              0
Coeur d'Alene R. 7/23/2011        0            0              0
Wolf Lodge Cr. 3/19 & 3/
  25/2011                         0            0              0
Fisher R. 4/27/& 4/28/2013        0            0              0
Flat Cr. 7/23/2011                0            0              0
St. Joe R. 3/18 & 3/26,
  2011, 4/5 & 4/8, 2012
  & 3/28/2013                    107           20             4
St. Joe R. Avery 9/4/2006

TABLE 3.--Sex chromosome diversity in the S. arcticum
complex at the Lower St. Joe River,  St. Maries, Benewah
Co., Idaho (combinational types are in italics and
percentages of females and males are in parentheses


              [X.sub.0]      [X.sub.0]#       [X.sub.IIL-2]
Date          [X.sub.0]    [X.sub.IIL-2]#     [Y.sub.IIL-2]

3/18,         85 (0.80)       19(0.18)#         2 (0.02)
  (n = 258)

4/5,          61 (0.84)      12 (0.16)#           0 (0)
  (n = 200)

3/28/2013     72 (0.69)      32 (0.31)#           0 (0)
  (n = 268)


                [X.sub.0]#       [X.sub.IIL-2]    [X.sub.IIL-2]#
Date            [Y.sub.0]#         [Y.sub.0]      [X.sub.IIL-3]#

3/18,           13 (0.09)#         13 (0.09)        25 (0.16)#
  (n = 258)

4/5,             7 (0.06)#         16 (0.13)         6 (0.05)#
  (n = 200)

3/28/2013       32 (0.20)#         25 (0.15)        15 (0.09)#
  (n = 268)


                 [X.sub.0]         [X.sub.0]
Date           [X.sub.IIL-3]    [X.sub.IIL-79]

3/18,            52 (0.34)         41 (0.27)
  (n = 258)

4/5,             44 (0.35)         43 (0.34)
  (n = 200)

3/28/2013        64 (0.39)         23 (0.14)
  (n = 268)


                [X.sub.2]          [X.sub.0]
Date          [X.sub.IIL-79]#  [X.sub.IIL-3 x 79]#

3/18,            7 (0.05)           1 (0.01)
  (n = 258)

4/5,             9 (0.07)           2 (0.02)
  (n = 200)

3/28/2013        4 (0.03)           1 (0.01)
  (n = 268)

Note: Combinational types are indicated with #.

TABLE 4.--Distribution of sex chromosome types within the
S. arcticum complex at the Kootenai River, Lincoln
County, Montana

              [female]              [male]
              [female]              [male]

              [X.sub.0]   [X.sub.0]   [X.sub.0]
Site          [X.sub.0]   [Y.sub.0]   [X.sub.IIL-9]

Kootenai R.   48          13           15


              [X.sub.0]        [X.sub.0]        [X.sub.0]
Site          [X.sub.IIL-13]   [X.sub.IIL-19]   [X.sub.IIS-15]

Kootenai R.   3                3                18


Site          [X.sub.IIL-9, IIS-15]

Kootenai R.   1

TABLE 5.--Sex-chromosome diversity within the S. arcticum complex at
Morrell Creek and at the Swan and Yaak rivers

                      [female]                      [male]
                      [female]                      [male]

              [X.sub.0]      [X.sub.0]      [X.sub.0]     [X.sub.0]
Site          [X.sub.0]   [X.sub.IIL-23i]   [Y.sub.0]   [X.sub.IIL-3i]

Morrell Cr.      10              0              2             1
Swan River        6              1              0             0
Yaak River        8              3              2             3


               [X.sub.0]     [X.sub.0]       [X.sub.0]
Site          [X.sub.IIL-   [X.sub.IIL-   [X.sub.IIL-22i]
                3i,23i]     3i,23i,24i]

Morrell Cr.        1             1               0
Swan River         0             3               1
Yaak River         0             0               1


               [X.sub.0]     [X.sub.0]       [X.sub.0]
Site          [X.sub.IIL-   [X.sub.IIL-   [X.sub.IIL-23i]
               22i,23i]      22i,24i]

Morrell Cr.        0             0               1
Swan River         1             1               0
Yaak River         0             0               1

TABLE 6.--Sex chromosome diversity within the Simulium arcticum
complex at Nason Creek, central Washington state


Site             [X.sub.0]      [X.sub.III/2]
               [X.sub.III/2]    [X.sub.III/2]

Nason Creek          2                13


Site             [X.sub.0]        [X.sub.0]
                 [Y.sub.0]      [Y.sub.III/2]

Nason Creek          3                19

TABLE 7.--Contrast between sex chromosome types at the Cle Elum River
(a S. saxosum site to the west), Little Prickly Pear Creek (a S.
arctcium s. s. site to the east) and the Coeur d'Alene River (a
combinational site)

Sex chromosome type    [X.sub.0]   [X.sub.0]   [X.sub.2]   [X.sub.0]
                       [X.sub.0]   [X.sub.2]   [X.sub.2]   [Y.sub.0]

  Cle Elum (1)             0           4          157          0
  Coeur d'Alene           301         138         26          110
  River (2)
  Little Prickly          801          0           0           0
    Pear Creek (3)

Sex chromosome type    [X.sub.2]   [X.sub.0]   [X.sub.2]
                       [Y.sub.0]   [Y.sub.3]   [Y.sub.3]

  Cle Elum (1)            115          0           0
  Coeur d'Alene           113         124         116
  River (2)
  Little Prickly           0          767          0
    Pear Creek (3)

(1) Shields and Kratochvil, 2011

(2) Shields, 2013 and this publication

(3) Shields, 2013
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Author:Shields, Gerald F.
Publication:The American Midland Naturalist
Article Type:Report
Geographic Code:1U8ID
Date:Jul 1, 2014
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