Good species behaving badly: Non-monophyly of black fly sibling species in the Simulium arcticum complex (Diptera: Simuliidae)

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Abstract

Mitochondrial based phylogenetic reconstructions often show deviations from species-level monophyly. We used the Simulium arcticum species complex (Diptera: Simuliidae) as a model system for interpreting non-monophyly in light of chromosomal data supporting species status of siblings. For cytogenetic identification of morphologically indistinguishable black fly sibling species, larvae must be preserved in Carnoy’s solution, a fixative known to degrade DNA. Consequently, we reconstructed phylogenetic relationships based on 12S, COII, cyt b, and ITS-1 gene sequences obtained from larvae sampled from presumed taxon-pure localities. As species composition at ‘taxon-pure’ sites may have changed at the time of sampling, we performed a second study that aimed to: (1) assess phylogenetic relationships among cytologically verified members of the S. arcticum species complex using COI and COII gene sequences; (2) determine whether useable genetic information could be gleaned from Carnoy’s fixed specimens; and (3) determine the extent to which Carnoy’s fixative degrades DNA over time. We consistently obtained genetic data from material stored in Carnoy’s solution for two to three months. Genetic analysis of samples fixed in Carnoy’s solution for up to six years indicates that larvae preserved for a maximum of five years can provide useable information for molecular analysis. Our preliminary and cytologically confirmed phylogenetic analyses demonstrate that mitochondrial DNA fails to resolve species-level monophyly of chromosomally distinct S. arcticum taxa. As results of analyses based on cytologically verified larvae mirror those of our preliminary study, we rule out imperfect taxonomy as the reason for species-level non-monophyly. Although we cannot confidently reject either inadequate phylogenetic information or incomplete lineage sorting as the cause of non-monophyly, the sharing of alleles between sympatric siblings suggests introgressive hybridization between taxa. We conclude that the patterns present in the S. arcticum phylogeny likely represent the initial stages of chromosome based sibling speciation.

Introduction

Mitochondrial DNA (mtDNA) offers rapidly evolving molecular markers for reconstructing phylogenetic relationships among closely related taxa. Yet, mtDNA analyses often show deviations from species-level monophyly; sequences from different species are more closely related to each other than to conspecific alleles. Funk and Omland (2003) surveyed 14 journals over 13 years and found that 23% of all studies evaluating mitochondrial gene trees showed species-level non-monophyly. Interestingly, 24% of these studies offered no discussion on the issue. Of the articles that acknowledged lack of monophyly, 50% attributed the problem to faulty taxonomy. Such casual dismissal overlooks potential information lying within gene trees exhibiting non-monophyly, such as inadequate phylogenetic signal, gene paralogy, incomplete lineage sorting of alleles, and/or introgressive hybridization between taxa. The latter two phenomena follow recent speciation events and can have important evolutionary implications (Funk and Omland, 2003). Given the significance of non-monophyly in mitochondrial gene trees, one must thoroughly examine this pattern in light of data indicating species-level monophyly. This type of investigation can provide insights into how speciation progresses from a micro- to macroevolutionary level.

Black flies (Diptera: Simuliidae) are ideal candidates for studying deviations from species-level monophyly. Simuliids are notorious for their structural homogeneity, and cytological analyses of larval salivary gland chromosomes reveal that morphologically defined taxa are often composites of chromosomally distinct sibling species. Chromosomes are inherited elements of the nuclear genome and rearrangements within them represent independent units of mutation (Dobigny et al., 2004). As such, chromosomal inversions have become accepted taxonomic and phylogenetic indicators in black flies (Rothfels, 1979, Rothfels, 1981, Rothfels, 1989). Several species complexes have been discovered in black flies through cytogenetic studies, including the Simulium arcticum (Adler et al., 2004, Shields and Procunier, 1982), S. damnosum (Dunbar, 1966, Vajime and Dunbar, 1975), S. venustum (Rothfels et al., 1978), S. verecundum (Rothfels et al., 1978), S. vittatum (Rothfels and Featherston, 1981), and Helodon onychodactylus (Newman, 1983) complexes. In general, reproductively isolated sibling species maintain unique chromosomal rearrangements (sex-linked and autosomal inversions) in sympatry with other closely related members of a particular species complex. Since sex chromosomes typically diverge first, cytological types (‘cytotypes’) distinguished only by fixed sex-linked inversions are hypothesized to represent the initial stages of the speciation process (Adler et al., 2004, Rothfels, 1979, Rothfels, 1989). Accordingly, cytogenetic studies provide a nuclear background from which to compare ecological and molecular data.

Ecological evidence tends to support the species status of siblings. For example, isolating mechanisms among sympatric sibling species include differential microhabitat preference and asynchronous emergence time (Adler and Kim, 1984, Brockhouse, 1985, Rothfels, 1989, Shields and Procunier, 1982, Shields et al., 2007a, Shields et al., 2007b). Unlike ecological studies, molecular analyses of black fly siblings are relatively scarce. Still, some trends are beginning to emerge from molecular data. For example, three independent studies found that length differences in internal transcribed spacer (ITS) loci of nuclear ribosomal RNA (rRNA) were useful for distinguishing cytologically defined types within the medically important S. damnosum species complex (Brockhouse et al., 1993, Kruger et al., 2000, Mustapha et al., 2005). Using cytogenetic data along with molecular sequences derived from the 16S rRNA and NADH dehydrogenase subunit 4 genes, Higazi et al. (2000) discovered a new S. damnosum sibling from the Nile region, in Sudan, that was similar to, but distinct from, two savannah-dwelling members of the complex. On the other hand, Xiong and Kocher (1993) showed that 16S rRNA alone is of limited utility for studies of siblings within the S. venustum and S. verecundum species complexes. Similarly, 16S rRNA and two fragments of the cytochrome c oxidase subunit I (COI) gene could not successfully distinguish S. crenobium from two other members of the S. vernum group (Ilmonen et al., 2009). Results from other studies using cytochrome c oxidase genes have been promising. From the analysis of 13 species representing four black fly genera, Pruess et al. (2000) concluded that COII successfully resolves species-level relationships, yet is of limited value at higher taxonomic levels. Similarly, the COI barcoding gene has proven useful for species identification of Nearctic black flies (Rivera and Currie, 2009). Using this gene, Rivera and Currie (2009) confirmed cryptic diversity, as previously revealed by cytological analyses. Furthermore, Pramual et al. (2005) used COI to describe geographic structure among populations of a single black fly species from Thailand. Finally, in reconstructing the evolutionary history of the subgenus Inseliellum, Joy and Conn (2001) showed that various genes support different portions of the tree. Specifically, the 12S gene resolved basal relationships, while COI was an important marker for recently diverged lineages.

Here, we use the Simulium arcticum species complex as a model system for interpreting species-level non-monophyly in mitochondrial phylogenies in light of chromosomal data supporting species status of siblings. The diverse S. arcticum species complex contains nine reproductively isolated sibling species and an additional 21 chromosomally distinct types that may, or may not represent valid species (Table 1). This spectacular example of chromosomal differentiation places the S. arcticum complex among the most species-rich complexes of black flies known, second only to the S. damnosum species complex of Africa (Adler and Crosskey, 2009). Simulium arcticum taxa are distributed throughout the Nearctic region, primarily in the Western Cordillera, from Alaska to Arizona (Adler et al., 2004). Although the S. arcticum complex occurs widely in western North America, a large portion of its biodiversity is concentrated in the Rocky Mountains of Montana, USA (Adler et al., 2004, Shields et al., 2007a, Shields et al., 2007b, Shields et al., 2009). Chromosome based phylogenetic reconstructions among S. arcticum siblings have not been forthcoming. These analyses recover monophyly of individuals representing a particular taxon, yet cannot resolve relationships between them. This is because S. arcticum taxa differ only in unique sex chromosome rearrangements, having no characteristics shared among them. Given a lack of morphological differentiation among S. arcticum members, along with concentrated taxonomic diversity in Montana, it is hypothesized that populations in the Rocky Mountains have experienced rapid chromosome based speciation as a result of recent glacial cycles (Pierce, 2003).

Morphologically indistinguishable black fly sibling species, identified only through banding pattern analysis of larval salivary gland chromosomes, poses a challenge for molecular studies. The protocol for cytogenetic analysis requires that larvae be fixed in Carnoy’s solution (1 part glacial acetic acid: 3 parts ethyl alcohol), which is known to degrade DNA (Koch et al., 1998, Post et al., 1993). In an effort to avoid this problem, we initially reconstructed phylogenetic relationships among S. arcticum siblings by sampling ethanol-fixed larvae from presumed taxon-pure localities (Shields et al., 2007a, Shields et al., 2007b; Shields, unpublished). However, given that species composition at ‘taxon-pure’ sites may have changed at the time of sampling, we could not rule out the possibility that instances of species-level non-monophyly were actually the result of misidentification. Consequently, we performed a second study that aimed to: (1) assess phylogenetic relationships among cytologically verified members of the S. arcticum species complex; (2) determine whether useable genetic information could be gleaned from Carnoy’s fixed specimens; and (3) determine the extent to which Carnoy’s fixative degrades DNA over time. Here, we present results from both our preliminary and cytologically confirmed studies in order to interpret the basis for non-monophyly of cytologically verified taxa within the S. arcticum complex.

Section snippets

Taxon sampling

We sampled four localities in central-southwestern Montana, USA, where pure populations of Simulium arcticum taxa were known to exist based on previous chromosomal surveys (Shields et al., 2007a, Shields et al., 2007b; Shields, unpublished). A total of 20 larvae representing four taxa were collected: cytotype IIL-10, individuals A–E: Upper Spring Creek, Fergus County; cytotype IIL-73,74, individuals A–E: Wise River, Beaverhead County; S. apricarium, individuals A–E: Jefferson River, Broadwater

Preliminary study based on larvae sampled from putatively pure populations

Relationships within the Simulium arcticum species complex were reconstructed based on: (1) larvae sampled from presumed taxon-pure localities and (2) 12S, COII, cyt b, and ITS-1 gene sequences. Sequence alignments consisted of 2281 positions. Of these characters, 12S, COII, cyt b, and ITS-1 made up 556, 729, 313, and 683 bases, respectively. We were unable to obtain COII sequence data for S. negativum 73. A total of 2157 molecular characters were excluded from MP analyses, 1860 of which were

Genetic information from Carnoy’s fixed samples

Previous studies suggest that useable genetic information cannot be obtained from insect larva fixed in Carnoy’s solution (Koch et al., 1998, Post et al., 1993). This fixative degrades DNA, yet is required to preserve entire chromosomes for cytogenetic studies. Molecular analyses of morphologically indistinguishable black fly sibling species, identified only through banding pattern analysis of larval salivary gland chromosomes, are particularly burdened by this predicament. Such studies require

Acknowledgments

This paper is dedicated to the memory of Mike Spironello, whose preliminary research on this subject provided the foundation on which the current paper is based. We thank J. Geiger and C. Blair for helpful advice concerning methodology. This research was supported by funding from the M. J. Murdock Charitable Trust (2003196; 2005233) to G.F. Shields; the James J. Manion Biology Fund of Carroll College; the Natural Sciences and Engineering Research Council of Canada, Discovery Grant to D.C.

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