A remnant of an incipient speciation event in the Simulium arcticum complex (Diptera: Simuliidae).
Black flies (Diptera: Simuliidae) provide an interesting case study of the role that chromosome change may play in the diversification process because the morphospecies of classical taxonomy almost always reveals itself as any number of sibling species when polytene chromosomes of larval salivary glands are analyzed (Rothfels, 1988). Sex-linked chromosomal inversions usually occur first in males and give rise to any number of cytogenetically differentiated types in sympatry (Rothfels, 1988). Shields and Procunier (1982) described four sibling species of the Simulium arcticum complex in Alaska, and Procunier (1984) described a fifth in western Canada based on possession of unique paracentric inversions in Y chromosomes. Adler et al. (2004) have formally named four of these five taxa based on other features of their biology. Full siblings of the complex include S. brevicercum (chromosomally IIL-standard), S. saxosum (IIL-2; IIL-2 refers to the unique sex-linked inversion on the long arm of chromosome II of this sibling), S. arcticum sensu stricto (IIL-3) and S. negativum (IL-3.4). The biology of the fifth taxon, S. arcticum IIL-1, has not been investigated in detail. Additionally, we have described 19 other types within the S. arcticum complex based on Y chromosome linkage of novel paracentric inversions in the long arm of chromosome II (Shields et al., 2007a, b; Shields et al., 2009). These types are initially referred to as cytotypes until their reproductive status and formal biology have been investigated. Investigation of species-specific sex chromosome types together with combinations of the two in sympatry provides a unique opportunity to document past evolutionary events. Specifically, is there evidence for "mating trials" between what have become full siblings, and, if so, are the various combinations of sex chromosome types in genetic equilibrium suggesting an incipient speciation event? A complete listing of taxa of the S. arcticum complex is given in Conflitti et al. (2010).
Our Mar. and Apr, collections in 2004 and 2005 at the Coeur d'Alene River, Kingston, Idaho indicated the presence of larvae of Simulium saxosum and S. arcticum s. s. as well as larvae possessing combinations of S. saxosum and S. arcticum s. s. sex chromosomes. This discovery was fortuitous since unlike most females of the S. arcticum complex, females of S. saxosum tend toward fixation of the inverted homozygote (IIL-2 i/i) while males are predominantly heterozygotes, IIL-2 st/i (Shields and Procunier, 1982; Adler et al., 2004). Alternatively, and like most other females of the S. arcticum complex, females of S. arcticum s. s. occur almost universally in the standard condition (IIL-st/st) while males are IIL-3 heterozygotes (IIL-3 st/i and IIL-2 st/st). This combination of X and Y chromosomes in larvae of the S. arcticum complex at the Coeur d'Alene River allowed individual chromosomal identification of not only all classes of males and females of both S. saxosum and S. arcticum s. s. but also of combinations of the two ([X.sub.0][X.sub.2], [X.sub.0][Y.sub.0], and [X.sub.2][Y.sub.3]).
Though the two species can be unequivocally identified only by analysis of their sex chromosomes (Shields and Procunier, 1982; Adler et al., 2004), the sympatric presence of Simulium saxosum and S. arcticum s. s. along with combinational types at the Coeur d'Alene River was unexpected since the described species have essentially exclusive geographic distributions and differences in other features of their biology. S. saxosum occurs generally in rocky flows of coastal regions of Alaska south through the Yukon, British Columbia, Washington State, Oregon and northern California (Shields and Procunier, 1982; Adler et al., 2004). Larvae develop in winter and pupae are present in Mar. (Adler et al., 2004). Alternatively, S. arcticum s. s. occurs generally in drainages of the eastern slopes of the Rocky Mountains from central Saskatchewan to southeastern British Columbia, western Montana, Wyoming and eastern Idaho (Fig. 1; and Shields and Procunier, 1982; Adler et al., 2004; Shields et al., 2007a, b; Shields et al., 2009). Larvae of S. arcticum s. s. are present throughout the year and development is triggered by increasing water temperatures in spring (Adler et al., 2004).
There are several interpretations for the cytogenetic diversity in sex chromosomes of taxa at the Coeur d'Alene River. First, Shields and Procunier (1982) observed that the IIL-2 inversion was autosomal in Simulium arcticum s. s. in Alaska. The presence of IIL-2/IIL-3 males, the absence of IIL-2 inversion homozygotes in males and the absence of IIL-3 inversion homozygotes in any larvae of the present study would rule out this possibility. Secondly, the combination of sex chromosome types at the Coeur d'Alene might be explained by interspecific hybridization between S. saxosum and S. arcticum s. s. (Rothfels, 1980; Procunier, 1989). In this case, sex chromosomes and autosomal polymorphisms should not be in genetic equilibrium and there should be differences in the extent of chromosome pairing and morphologies of chromocenters when putative parental and combinational types are compared. Thirdly, there is the possibility that the population at the Coeur d'Alene is panmictic and with all types represented nearly equally year after year. In this case, the population at the Coeur d'Alene may be the putative ancestor to present day S. saxosum and S. arcticum s. s. and the in situ remnant of a past incipient speciation event. If so, we might expect all sex chromosome types to occur year after year and that they and autosomal polymorphisms would be in equilibrium in 2009 and in 2010. We might also expect chromosome pairing and chromocenter morphologies not to differ when species specific and combinational types are compared.
[FIGURE 1 OMITTED]
We made more extensive collections at the Coeur d'Alene site in 2009 and 2010 to (1) verify the presence and frequencies of larvae identified in earlier collections, (2) test for genetic equilibrium among sex chromosomes and the most abundant autosomal polymorphism, IS-1, (3) determine the extent of chromosome pairing and morphology of chromocenters among parental and intermediate types and (4) determine the geographic extent of these types by analyzing larvae from three nearby sites to the west and four nearby sites to the east. Since Adler et al. (2004) described Simulium saxosum and S. arcticum s. s. as good species, we hypothesized that the combinational types at the Coeur d'Alene arose though interspecific hybridization.
MATERIALS AND METHODS
We used conventional methods of collection (Shields and Procunier, 1982) and identification of larvae (Currie, 1986), the Feulgen method of Rothfels and Dunbar (1953) to stain polytene chromosomes and gonads of larvae and the standard chromosome maps for the Simulium arcticum complex (Shields and Procunier, 1982) to determine chromosomal variation. Gonads were studied to provide an on-slide identifier of sex and determine the latest stage of meiosis and the presence or absence of B, supernumerary chromosomes. Some slides were made permanent so that photographs of the various chromosome morphs could be made. Permanent slides also serve as documentation for future reference. Slides were made permanent by freezing them at -80 C for at least 1 h, flicking off the cover slip with a razor blade, clearing the slide in EtOH, and remounting a new cover slip over Euparal.
We particularly analyzed the long arm of chromosome II for variation in banding sequence. We maximized our collection efforts in 2009 and 2010 so that cohorts of roughly equal sample size could be compared for the presence and frequency of each of the seven sex chromosome genotypes. We then used a contingency analysis and the [chi square] statistic to determine if the distribution of genotypes differed from 2009 to 2010. We used the Hardy-Weinberg equation and the [chi square] test to determine (1) if larvae collected in 2009 and in 2010 were each in genetic equilibrium for sex chromosomes and for the IS-1 autosomal polymorphism.
We used the method of Brockhouse and Adler (2002) to determine if 10 combinational types exhibited less polytene chromosome pairing than did 10 putative parental types from prepared slides from a 5 Apr. 2009 collection at the Coeur d'Alene. We digitally photographed well-spread nuclei from each of the ten combinational types and compared them to those of ten sibling types. From enlarged photographs, we determined the total complement length and calculated percentages of paired and unpaired segments, excluding heterozygotic inversions. Extent of pairing on the photographs was verified by reference to those regions on the actual chromosomes preserved on permanent slides.
We averaged the two-dimensional areas (greatest length and width via an ocular micrometer) of the chromocenters (three amplified centric regions) from 10 Simulium saxosum/S. arcticum s. s. and 10 combinational types from the Coeur d'Alene and used a standard t-test to determine significance. Finally, we compared the polytenes of larvae at the Coeur d'Alene River to those at seven nearby sites to determine if the complex discovered at the Coeur d'Alene occurred anywhere else (Table 1).
Three hundred seventy of the 382 larvae of the Simulium arcticum complex analyzed from the Coeur d'Alene River site were S. saxosum, S. arcticum s. s. or combinations of the two (Table 2; Figs. 2-4). All types were found in most collections, particularly the larger ones, suggesting that the types have been present in nearly equal frequencies at least since 2004. Since no larvae of the S. arcticum complex were observed in May and Aug. collections at the Coeur d'Alene River (Table 2), we assumed that the cohort there has a single generation per year.
Sex chromosomes of 2009 and 2010 populations at the Coeur d'Alene were each in genetic equilibrium ([X.sup.2] = 3.40, d.f. = 6, P = 0.757 and [X.sup.2] = 1.05, d.f. = 6, P = 1.0, respectively, Table 3). Similarly, the distribution of the IS-1 autosomal polymorphism was also in equilibrium for both years ([X.sup.2] = 2.82, d.f. = 2, 0.20 < P < 0.30 and [X.sup.2] = 0.64, d.f. = 2, 0.70 < P < 0.80, respectively, Table 4). Moreover, the distribution of sex chromosome genotypes for 2009 and for 2010 was very similar ([X.sup.2] = 3.8, d.f. = 6, P = 0.704; Table 5). Extents of chromosome pairing among sibling ([bar.x] = 13.9%, SD = 0.076) and combinational ([bar.x] = 14.9%, SD = 0.113) types were not significantly different (t = -0.25, d.f. = 18, P = 0.805). Likewise, the two-dimensional areas of chromocenters in sibling ([bar.x] = 754.9, SD = 279.9) and combinational ([bar.x] = 758.7, SD 299.6) types were not significantly different (t = -0.029, d.f. = 18, P = 0.98).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
The nearest pure Simulium saxosum site is at the Cle Elum River 356 km. to the west (Table 6) while the nearest predominantly S. arcticum s. s. sites are at the St. Regis River (Haugen and St. Regis), 131 and 170 km. to the east, respectively (Table 6). Surprisingly, the two sites closest to the Coeur d'Alene have larvae that are neither S. saxosum nor S. arcticum s. s. but rather are S. arcticum IIL-9 (Shields et al., 2007b) at the Spokane River (Table 6) and a new cytotype, IIL-21, at Latah Creek (Fig. 5 and Table 6). No larvae of the S. arcticum complex were observed at the South Fork of the Coeur d'Alene River.
[FIGURE 5 OMITTED]
There are three possible explanations for the diversity of sex chromosome types at the Coeur d'Alene River. First, sex chromosomes of Simulium saxosum may be operating autosomally in S. arcticum s. s. and vice-versa. Second, the diversity may be the result of hybridization between S. saxosum and S. arcticum s. s. Third, the population may be panmictic and represent the remnant of an incipient speciation event that gave rise to S. saxosum to the west and to S. arcticum s. s. to the east.
Autosomal polymorphisms.--Shields and Procunier (1982) stated that the IIL-2 inversion occurs autosomally in the IIL-3 sibling (Simulium arcticum s. s.) in Alaska. Thus, it is possible that the IIL-3 types are autosomal in S. saxosum and that IIL-2 types are autosomal in S. arcticum s. s. However, the absence of IIL-2 inversion homozygotes in males and the absence of IIL-3 inversion homozygotes in any larvae of the present study suggest that neither the IIL-2 nor the IIL-3 inversions is operating autosomally in the population at the Coeur d'Alene.
Shields and Procunier (1982) also noted the presence of two X chromosomes and two Y chromosomes in the IIL-2 sibling (Simulium saxosum) in Alaska, for which both the X and Y could be standard or inverted for IIL-2. However, we never observed X2Y2 males in the present study. Moreover, IIL-2/IIL-3 males were among the most numerous types at the Coeur d'Alene; they were never seen in Alaska (Shields and Procunier, 1982). Thus, we rule out the possibility that alternative types are acting autosomally.
Interspecific hybridization.--It is possible that sex chromosome types of Simulium saxosum and S. arcticum s. s. and combinations of them at the Coeur d'Alene are the result of hybridization. However, there is no evidence in the present study to support hybridization. Specifically, genetic equilibrium for sex chromosomes and equilibrium for the IS-1 autosomal polymorphism in both 2009 and 2010 and lack of differences in pairing and chromocenter morphologies all argue against a hybridization event.
We recognize that types analyzed from two different dates (3/15 and 4/3) in 2009 might affect tests of equilibrium since types may change over time. However, if types changed over time it seems likely that disequilibrium would result. This was not the case in the present study. Additionally, no significant differences in either the distribution of sex chromosome types or the distribution of the IS-1 autosomal polymorphism were observed among the largest sample of 2010.
We recognize that comparisons of chromosome pairing may not be totally informative at the Coeur d'Alene River. Rothfels and Nambiar (1981) used reduced chromosome pairing in putative hybrids relative to parental types of Prosimulium multidentatum and P. magnum, two morphologically distinct species, in New York state to argue for the presence of hybrids. Support for a hybridization event based on lack of pairing in putative hybrids assumes tight pairing of chromosomes in parental taxa. However, Simulium saxosum at the Cle Elum River exhibits reduced pairing (22.0% pairing, analysis of three individuals, data not shown) relative to taxa having complete pairing. Also, the extent of chromosome pairing may be influenced by water temperature. At Waterford, Ontario, the IIIL-1 sibling of S. vittatum has loose pairing in summer but tight pairing the rest of the year (Rothfels and Featherston, 1981). Thus, lack of significant differences in pairing of types at the Coeur d'Alene may not be totally informative though all of our analyses were based on spring samples.
A panmictic population.--The data gathered here suggest that the population of the Simulium arcticum complex at the Coeur d'Alene River is panmictic and is characterized by an X chromosome that is either st or 2 and a Y chromosome that is either st or 3. Relatively equal frequencies of all sex chromosome types in 2009 and 2010 and genetic equilibrium for those types and for the distribution of the IS-1 autosomal polymorphism for both years indicate that this population is in genetic equilibrium at the Coeur d'Alene. We, therefore, reject our hypothesis that the sex chromosome complexity at the Coeur d'Alene was caused by hybridization.
Rothfels (1980, 1989 ) proposed a "chromosome model of speciation" in simuliids in which initial "mating trials" might occur between cytological entities having polymorphic sex chromosomes. Coadaptation of polymorphic sex chromosomes in pairs may occur followed by reinforcement through assortative mating and slight selective advantage. Initial lineage splitting in sympatry is based only on polymorphism in X and Ychromosomes, as is the case in the Simulium arcticum complex at the Coeur d'Alene. Moreover, recombination would not exist between sex chromosomes, or in this case, in regions of inversions containing the sex-determining gene or genes, allowing for the initiation of isolating barriers to reproduction (Rothfels, 1989) and Hirai et al. (1994). Possibly, the distribution of types at the Coeur d'Alene River are examples of these previously hypothesized "mating trials."
Simulium saxosum and S. arcticum s. s are incipient species?--The presence of sex chromosome types of S. saxosum, S. arcticum s. s. and combinations of them in equilibrium may suggest that the Coeur d'Alene population is a remnant of the incipient speciation event. It is possible that the population at the Coeur d'Alene gave rise to present day S. saxosum to the west and to S. arcticum s. s. to the east. That is, classic S. saxosum in coastal regions of the Northwest may have retained the [X.sub.2][X.sub.2]/[X.sub.2][Y.sub.0] sex-determining system of the Coeur d'Alene cytoform while losing the [X.sub.-0][X.sub.0]/[X.sub.0][Y.sub.3] sex-determining system of S. arcticum s. s. Correspondingly, S. arcticum s. s. of the Rocky Mountain regions to the east may have retained the [X.sub.0][X.sub.0]/[X.sub.0][Y.sub.3] sex-determining system while losing the [X.sub.2][X.sub.2]/[X.sub.2][Y.sub.0] system.
Rothfels and Featherston (1981) used sex chromosome identity and the frequency and distribution of autosomal polymorphisms to suggest that Simulium vittatum (Zett.) is composed of two sibling species, IIIL-1 and IS-7. Unlike the situation at the Coeur d'Alene, the two siblings of S. vittatum occur sympatrically in much of their geographic ranges, and Rothfels and Featherston (1981) argued that the two siblings were derived from a precursor population in the Great Lakes area since it seemed unlikely that they would hybridize only in the region of the Great Lakes and nowhere else. Support for secondary contact and hybridization in the case of S. vittatum would require the reversal of two well differentiated sex chromosome systems and the difficulty in explaining the sharing of autosomal polymorphisms in areas of allopatry (Rothfels and Featherston, 1981).
Duncan et al. (2004) used randomly amplified polymorphic DNA on eastern and western populations of Simulium vittatum, and, in contrast to Rothfels and Featherston (1981), concluded that IS-7 vittatum evolved to the west and spread eastward, while IIIL-1 vittatum originated later to the east from an entity similar to IS-7 but lost its differentiated sex chromosomes and spread westward. This interpretation implies a reversal of differentiated sex chromosome systems into undifferentiated ones involving the loss of sex linkage of an X chromosome inversion (IS-7) and the derivation of differentiated Y chromosome (IIIL-1). Possibly, sex chromosome systems can be lost and differentially gained to give rise to derived cytoforms as may be the case at the Coeur d'Alene.
Support for the hypothesis that Simulium saxosum and S. arcticum s. s. may be incipient species may be strengthened by our own recent molecular data that indicate that chromosomally identified larvae of S. saxosum and S. arcticum s. s. are not reciprocally monophyletic in phylogenetic trees based on comparisons of mitochondrial and nuclear DNA sequences (Conflitti et al., 2010). This molecular study indicates that variation in DNAs has not yet become fixed in each taxon. This might suggest (1) that sex chromosome variation may occur prior to fixation of molecular variation and (2) that S. saxosum and S. arcticum s. s. are incipient species. If this is indeed the case, genetic equilibrium for sex chromosomes and for the IS-1 autosomal polymorphism, as shown here, is expected.
Our interest in the reproductive compatibilities of various taxa of the Simulium arcticum complex has led us to test equilibrium frequencies of taxon pairs where they occur sympatrically. If unique Ychromosome inversions are important in the differentiation process in the complex, then determination of the reproductive compatibilities of these taxa in sympatry should be revealing. In these cases, we have sought locations where at least two taxa of the complex are abundant, where heterozygosities of autosomal polymorphisms, such as IS-1, are high enough to obtain accurate tests of equilibrium and where chromosome banding was of good visual quality. Accordingly, in Montana we have determined that S. apricarium and S. arcticum s. s. at Little Prickly Pear Creek and S. negativum and S. arcticum IIL-9 at the Blackfoot River are reproductively isolated (Shields et al., 2007b) and that two new cytotypes (IIL-9 and IIL-19) at Rock Creek (Shields et al., 2007b) and S. arcticum s. s. and a new cytotype (IIL-22) at the Clearwater River (Shields et al., 2010) are not reproductively isolated. The importance of the situation at the Coeur d'Alene River is that all possible combinations of sex chromosomes of S. saxosum and S. arcticum s. s. and their combinational types are present and that these types are in genetic equilibrium. That the situation at the Coeur d'Alene is rare and, therefore, significant is the fact that we found only two combinational types, both IIL-3/IIL7 males, among 5042 larvae analyzed in the four aforementioned studies of reproductive status. We are not aware of other situations where combinational sex chromosome types are abundantly present with sibling specific types.
Observations on the distribution of the IS-1 autosomal polymorphism and on B chromosomes at sites near the Coeur d'Alene appear not to be informative regarding the derivation of types. The IS-1 inversion has a heterozygosity of 0.09 at the Coeur d'Alene River. Similar heterozygosities for IS-1 are seen at the Spokane River (0.21) to the west and at the St. Regis River (0.10) to the east. Two of the 215 male Simulium arcticum s. s. larvae analyzed at the Coeur d'Alene each had two acrocentric B chromosomes. The S. arcticum IIL-9 population at the Spokane River is replete with Bs (0.85 Bs/male larva); Bs also occur in male larvae at Latah Creek (0.06/male larva) and at the St. Regis River (0.02/male larva) though in lower numbers.
There is little evidence that the population discovered at the Coeur d'Alene is widespread. Only five males at the St. Regis River and one male at Latah Creek were IIL-2 st/i types. Moreover, only one male at St. Regis was a IIL-2/IIL-3 heterozygote. Our sampling of sites near the Coeur d'Alene has been oriented in an east-to-west direction along U.S. Interstate Highway 90 for convenience of travel and collection. However, given the known geographic distributions of Simulium saxosum and S. arcticum s. s. (Adler et al., 2004; Fig. 1) areas of potential sympatry may run north and south. It is possible that additional populations like those at the Coeur d'Alene might be discovered with more wide spread sampling.
A rather new area of investigation centers on hybridization between diploid species without increase in chromosome number (Reiseberg, 1997). Such an event may lead to establishment of new lineages via the process of recombinational speciation in which a novel genotype is formed that is homozygous for several chromosomal rearrangements and results in a new, fertile and true-breeding lineage (Grant, 1981; Reiseberg, 1997). Recombinational speciation is not well understood since it probably occurs infrequently and is, therefore, difficult to detect (Reiseberg, 1997; Coyne and Orr, 2004). The population at the Coeur d'Alene River may be the remnant of such an event. As Coyne and Orr (2004) stress, it is important to investigate and understand processes that occur prior to and during a speciation event. A posteriori accounts of speciation are plentiful in the scientific literature, but processes that initially promote differentiation are difficult to detect and explain in detail. Continued study of the black fly larvae at the Coeur d'Alene site and nearby drainages with particular emphasis on all features of their biology could be informative. Moreover, we are currently comparing DNA microsatellites of chromosomally identified larvae from the Coeur d'Alene River, the Cle Elum River (pure Simulium saxosum), and the Clearwater River (essentially a S. arcticum s. s. site). Analytical protocols that determine the direction and rate of gene flow among these populations are being employed. Possibly, these new molecular data will further clarify the genetic situation at the Coeur d'Alene River.
Acknowledgments.--This research was supported by grants from the M.J. Murdock Charitable Trust (#2003196 and #2005233) to Shields. Shields' research time was supported by the James J. Manion Endowed Chair of Biological Sciences Fund at Carroll College. Space and equipment were provided by the Department of Natural Sciences at Carroll. John and Pat Shields helped with collections. John Addis of Carroll College reviewed an earlier draft of this manuscript and Grant Hokit, also of Carroll, helped with statistical analyses. We have received extensive input on this study from Prof. Peter Adler of Clemson University for which we are grateful. Dr. William S. Procunier of Centennial College, Toronto, Canada reviewed the present draft.
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SUBMITTED 4 DECEMBER 2009
ACCEPTED 31 MARCH 2011
GERALD F. SHIELDS (1) AND MICHAEL J. KRATOCHVIL
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: email@example.com
TABLE 1.--Collection information for the nine sites included in this study Geographic Site name Location coordinates Cle Elum River Kittitas Co., Wa. 47[degrees] 10'30"N, 121[degrees] 01'40'W Latah Creek Spokane Co., Wa. 47[degrees] 33'50"N, 117[degrees] 30'20'W Spokane River Spokane Co., Wa. 47[degrees] 41'50"N, 117[degrees] 03'20'W Coeur d'Alene River Shoshone Co., Id. 47[degrees] 34'10"N, 116[degrees] 16'25'W S. Fork Coeur d'Alene R. Shoshone Co., Id. 47[degrees] 34'10"N, 116[degrees] 10'00'W St. Regis R., Haugen Mineral Co., Mt. 47[degrees] 24'00"N, 115[degrees] 22'15'W St. Regis River, St. Regis Mineral Co., Mt. 47[degrees] 18'00"N, 115[degrees] 10'00'W Clearwater River Missoula Co. Mt. 47[degrees] 00'03"N, 113[degrees] 22'94'W Rock Creek Missoula Co. Mt. 46[degrees] 40'28"N, 113[degrees] 40'10'W Distance from Coeur d'Alene Site name R. (km.) Collections Cle Elum River 356 W 3 Latah Creek 60 W 4 Spokane River 31 W 1 Coeur d'Alene River 0 11 S. Fork Coeur d'Alene R. 8 E 2 St. Regis R., Haugen 131 E 2 St. Regis River, St. Regis 170 E 1 Clearwater River 196 E 3 Rock Creek 203 E 16 TABLE 2.--Distribution of sex chromosomes among Simulium saxosum and S. arcticum s. s. and their combinational types (italics) at the Coeur d'Alene River, Kingston, Shoshone County, Idaho Date [X.sub.0][X.sub.0] [X.sub.0][X.sub.2] 3/11/10 (a, b) 14 19 3/13/2010 16 11 3/14/05 (c) 3 3 3/15/09 (d) 3 8 4/3/2009 8 4 4/5/09 (e) 12 15 4/8/2005 7 5 4/11/04 (f) 3 1 5/1/2009 no S. 5/29/2009 no S. 8/26/2010 only S. -- -- Total 66 66 Date [X.sub.2][X.sub.2] [X.sub.0][Y.sub.0] 3/11/10 (a, b) 5 18 3/13/2010 4 12 3/14/05 (c) 1 1 3/15/09 (d) 2 8 4/3/2009 2 3 4/5/09 (e) 7 6 4/8/2005 2 2 4/11/04 (f) 0 0 5/1/2009 arcticum# larvae 5/29/2009 arcticum# larvae 8/26/2010 vittatum# larvae -- -- Total 23 50 Date [X.sub.2][Y.sub.0] [X.sub.0][Y.sub.3] 3/11/10 (a, b) 9 15 3/13/2010 20 9 3/14/05 (c) 2 2 3/15/09 (d) 6 14 4/3/2009 7 4 4/5/09 (e) 5 5 4/8/2005 1 1 4/11/04 (f) 0 3 5/1/2009 5/29/2009 8/26/2010 -- -- Total 50 53 Date [X.sub.2][Y.sub.3] Total 3/11/10 (a, b) 20 100 3/13/2010 13 85 3/14/05 (c) 5 17 3/15/09 (d) 8 49 4/3/2009 9 37 4/5/09 (e) 5 55 4/8/2005 2 20 4/11/04 (f) 0 7 5/1/2009 5/29/2009 8/26/2010 -- -- Total 62 370 (a) An additional male was a IIL-7 heterozygote while another male was a IIL-9 heterozygote (b) An additional female was a IIL-7 heterozygote while two males and a female were IIL-90 (c) An additional male was a IIL-3 (st/st/i) triploid (d) An additional male was a IIL-3 (st-i-i) triploid while two additional males were IIL-9 heterozygote (e) An additional male and female were IIL-20 heterozygotes (f) Two additional IIL-3males each had two acrocentric B chromosomes Note: Distribution of sex chromosomes among Simulium saxosum and S. arcticum s. s. and their combinational types is indicated with #. TABLE 3.--Tests of random assortment of sex chromosomes [X.sub.0], [X.sub.2], [Y.sub.0] and [Y.sub.3] in the Coeur d'Alene River population of the Simulium arcticum complex for 2009 and 2010 2009 (n = 141) Frequency of [X.sub.0] [X.sub.2] [Y.sub.0] [Y.sub.3] 0.60 0.40 0.44 0.56 Classes [X.sub.0][X.sub.0] [X.sub.0][X.sub.2] Obs. 23 27 Exp. 22 29.3 Classes [X.sub.2][X.sub.2] [X.sub.0][Y.sub.0] Obs. 11 17 Exp. 9.8 21.1 Classes [X.sub.2][Y.sub.0] [X.sub.0][Y.sub.3] Obs. 18 23 Exp. 14.1 26.9 Classes [X.sub.2][Y.sub.3] Obs. 22 Exp. 17.9 [X.sub.0] = 3.40, d.f. = 6, P = 0.757 2010 (n = 185) Frequency of [X.sub.0] [X.sub.2] [Y.sub.0] [Y.sub.3] 0.65 0.35 0.51 0.49 Classes [X.sub.0][X.sub.0] [X.sub.0][X.sub.2] Obs. 30 30 Exp. 29.0 31.7 Classes [X.sub.2][X.sub.2] [X.sub.0][Y.sub.0] Obs. 9 30 Exp. 8.3 27.5 Classes [X.sub.2][Y.sub.0] [X.sub.0][Y.sub.3] Obs. 29 24 Exp. 31.5 26.5 Classes [X.sub.2][Y.sub.3] Obs. 33 Exp. 30.5 [X.sub.2] = 1.05, d.f. = 6, P = 1.0 TABLE 4.--Tests of Hardy-Weinberg equilibrium for the distribution of the IS-1 autosomal polymorphism among sex chromosome types at the Coeur d'Alene River in 2009 and 2010 2009 [X.sub.0][X.sub.0] [X.sub.0][X.sub.2] IS-1 st/st 13 14 IS-1 st/i 1 0 IS-1 i/i 0 1 2009 [X.sub.2][X.sub.2] [X.sub.0][Y.sub.0] IS-1 st/st 3 9 IS-1 st/i 1 1 IS-1 i/i 0 0 2009 [X.sub.2][Y.sub.0] [X.sub.0][X.sub.3] IS-1 st/st 10 10 IS-1 st/i 1 0 IS-1 i/i 0 0 2009 [X.sub.2][X.sub.3] IS-1 st/st 11 IS-1 st/i 2 IS-1 i/i 0 [X.sub.2] = 2.82, d.f. = 2,0.20 < P < 0.30 2010 [X.sub.0][X.sub.0] [X.sub.0][X.sub.2] IS-1 st/st 19 23 IS-1 st/i 3 2 IS-1 i/i 0 0 2010 [X.sub.2][X.sub.2] [X.sub.0][Y.sub.0] IS-1 st/st 7 27 IS-1 st/i 1 3 IS-1 i/i 0 0 2010 [X.sub.2][Y.sub.0] [X.sub.0][X.sub.3] IS-1 st/st 27 22 IS-1 st/i 0 4 IS-1 i/i 0 0 2010 [X.sub.2][X.sub.3] IS-1 st/st 30 IS-1 st/i 2 IS-1 i/i 0 [X.sub.2] = 0.64, d.f .= 2,0.70 <P < 0.80 TABLE 5--Contingency analysis determining whether the distribution of sex chromosome types was the same for 2009 and 2010 [X.sup.0][X.sup.0] [X.sup.0][X.sup.0] 2009 Obs. 23 27 Exp. 22.9 24.7 2010 Obs. 30 30 Exp. 30.1 32.3 [X.sup.0][X.sup.0] [X.sup.0][X.sup.0] 2009 Obs. 11 17 Exp. 8.65 20.3 2010 Obs. 9 30 Exp. 11.3 26.7 [X.sup.0][X.sup.0] [X.sup.0][X.sup.0] 2009 Obs. 18 23 Exp. 20.3 20.3 2010 Obs. 29 24 Exp. 26.7 26.7 [X.sup.0][X.sup.0] 2009 Obs. 22 Exp. 23.8 2010 Obs. 33 Exp. 31.2 [X.sup.2] = 3.8, d.f. = 6, P = 0.704 TABLE 6.--Comparisons of sex chromosome types at the Cle Elum River, Spokane River and Latah Creek west of the Coeur d'Alene (top three rows) and at the St. Regis River, Rock Creek and Clearwater River east of the Coeur d'Alene (bottom three rows) * Location [X.sub.0][X.sub.0] [X.sub.0][X.sub.2] Cle Elum (a) 0 4 Spokane (b) 14 0 Latah (c) 85 0 Coeur d'Al 66 66 St. Regis 3 5 Rock Cr. 621 0 Clear-water 700 0 Location [X.sub.2][X.sub.2] [X.sub.0][Y.sub.0] Cle Elum (a) 157 0 Spokane (b) 0 0 Latah (c) 0 10 Coeur d'Al 23 50 St. Regis 0 15 Rock Cr. 0 24 Clear-water 0 0 Location [X.sub.2][Y.sub.0] [X.sub.0][Y.sub.3] Cle Elum (a) 115 0 Spokane (b) 0 1 Latah (c) 1 0 Coeur d'Al 50 53 St. Regis 0 47 Rock Cr. 0 37 Clear-water 0 903 Location [X.sub.2][Y.sub.3] [X.sub.0][Y.sub.9] Cle Elum (a) 0 0 Spokane (b) 0 61 Latah (c) 0 0 Coeur d'Al 62 1 St. Regis 1 0 Rock Cr. 0 282 Clear-water 0 4 Location [X.sub.0][Y.sub.19] [X.sub.0][Y.sub.21] Cle Elum (a) 0 0 Spokane (b) 0 0 Latah (c) 0 83 Coeur d'Al 0 0 St. Regis 0 0 Rock Cr. 380 0 Clear-water 1 0 Location [X.sub.0][Y.sub.22] Cle Elum (a) 0 Spokane (b) 0 Latah (c) 0 Coeur d'Al 0 St. Regis 0 Rock Cr. 0 Clear-water 222 (a) The centromere dimorphism (Ce = enhanced, Ct = thin), IS-1 autosomal polymorphism (22.8% heterozygosity) and B chromosomes observed in Simulium saxosum from Alaska (Shields and Procunier, 1982) were not observed in S. saxosum at the Cle Elum River (b) Sixteen larvae were ISI heterozygotes, one [male] was a IS-1 homozygote. Four of 14 [female][female] were IIL20 heterozygotes as were five of the 62 [male][male]. Half (n = 23 of 46) of the [male] larvae with active meioses had acrocentric B chromosomes. Of these, seven act had two Bs, three had three and one had four (c) Twenty-three of the 68 [female][female] and 22 of 44 [male][male] were IL-1 heterozygotes. Each of three [male][male] had acrocentirc B chromosomes while a fourth [male] had two Bs. An additional [female] was [X.sub.2][Y.sub.21] * Values for Rock Creek and the Clear-water River are from Shields et al. 2007b and from Shields et al. 2010, respectively
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|Author:||Shields, Gerald F.; Kratochvil, Michael J.|
|Publication:||The American Midland Naturalist|
|Date:||Oct 1, 2011|
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