Molecular phylogenetic analysis of nycteribiid and streblid bat flies (Diptera: Brachycera, Calyptratae): Implications for host associations and phylogeographic origins

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Abstract

Bat flies are a small but diverse group of highly specialized ectoparasitic, obligatory bloodsucking Diptera. For the first time, the phylogenetic relationships of 26 species and five subfamilies were investigated using four genes (18S rDNA, 16S rDNA, COII, and cytB) under three optimality criteria (maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference). Tree topology tests of previous hypotheses were conducted under likelihood (Shimodaira–Hasegawa test). Major findings include the non-monophyly of the Streblidae and the recovery of an Old World- and a New World-Clade of bat flies. These data ambiguously resolve basal relationships between Hippoboscidae, Glossinidae, and bat flies. Recovered phylogenies resulted in either monophyly (Bayesian approach) or paraphyly (MP/ML topologies) of the bat flies, thus obscuring the potential number of possible associations with bats throughout the history of this group. Dispersal-vicariance analysis suggested the Neotropical region as the possible ancestral distribution area of the New World Streblidae and the Oriental region for the Old World bat flies. The genes examined show conflicting support across the nodes of the tree, particularly in the basal positions. Additionally, there is poor character support among all genes for the nodes associated with early hippoboscoid diversification. This results in extremely short basal branches, adding support to the idea of a rapid radiation among the four major groups of Hippoboscoidea.

Introduction

Bat flies are a group of highly specialized Diptera that are obligate ectoparasites on bats. Only the Old World genus Ascodipteron shows true female neosomy, and burrows into the skin of the host, thus being referred to as “endoparasitic” by some authors. Currently, 520 species are described (Húrka and Soós, 1986, Maa, 1989), which makes bat flies the most species-rich group among the calypterate Diptera associated with mammals. Two families are commonly recognized: Nycteribiidae and Streblidae (Marshall, 1981); however, in the Biosystematic Database of World Diptera streblid and nycteribiid bat flies are reported as part of the Hippoboscidae (http://www.sel.barc.usda.gov/Diptera/biosyst.htm). Bat flies are considered as part of the Hippoboscoidea, a calypterate superfamily encompassing several obligatory bloodsucking groups, including the medically important Glossinidae (tse-tse flies) and the Hippoboscidae, which feed on birds and mammals. Although both bat fly families show reduced compound eyes, no ocelli, a spider-like orientation of their legs, and adenotrophic vivipary (Meier et al., 1999), some of the characters that have led to the classification of two distinct bat fly families are the peculiar wing morphology with extensively membranous abdomen in the Streblidae, and the complete wing loss, the backwardly folded head, and dorsally inserted legs in the Nycteribiidae (McAlpine, 1989).

In addition to these morphological differences, the geographical and climatic distributions of both the families differ considerably. Streblidae are largely confined to subtropical and tropical climates and have a worldwide distribution, covering all biogeographic regions. However, Old- and New world faunae have no taxa in common (Kim and Adler, 1985). In addition to subtropical and tropical regions, Nycteribiidae occur in temperate climate, although their greatest diversity is in the Old World. Kim and Adler (1985) hypothesized that the center of origin for the Nycteribiidae might have been the Malaysian subregion (Oriental region) due to their high species diversity in this area.

Generally, bat flies are regarded as being not very host specific since most bat species exhibit high degrees of spatial overlap on geographic and habitat scales, and additionally often roost in dense colonies of conspecific individuals. Thus, there are ample opportunities for potential reassociations to hosts. However, it has been noted that bat fly assemblages may exhibit remarkable host specificity on all taxonomic scales (Patterson et al., 1999, Wenzel and Tipton, 1966). According to Marshall (1981) and Kim and Adler (1985), this is particularly obvious for the genus Megastrebla and the subfamily Cyclopodiinae, both exclusively associated to megachiropteran bats.

Bat flies are among the most specialized of all Diptera, and their highly specialized features have resulted in a confusing nomenclature. Earlier taxonomic work established two subfamilies of Streblidae: Streblinae and Nycteriboscinae [all New- and Old World taxa other than Strebla sensu Speiser] (Speiser, 1900). Later Jobling (1936) proposed the Trichobiinae (including Nycterophilia ) and reduced the family Ascodipteridae to a subfamily level. Wenzel et al. (1966) proposed an additional subfamily, Nycterophiliinae, for the New World genus Nycterophilia. Most recently, five streblid subfamilies, Nycteriboscinae, Ascodipterinae, Trichobiinae, Streblinae, and Nycterophilinae, are recognized. Maa (1965) renamed the Nycteriboscinae to Brachytarsininae, as he regarded the type genus as a junior synonym. This is not recognized by all authors. Within Nycteribiidae, there are currently three subfamilies Nycteribiinae, Cyclopodiinae (Theodor, 1967), and Archinycteribiinae (Maa, 1971).

One main evolutionary trend within the hippoboscoid lineages is bloodsucking, which spurred the potential for convergent evolution of reduced and/or specialized features, and has consequently led to several contradicting classification schemes. Griffith (1972) challenged the common division of bat flies into two distinct families, and includes the Streblidae, Nycteribiidae, and Hippoboscidae sensu strictu as subfamilies within the Hippoboscidae sensu latu, based on several shared characters concerning the 6th tergum, the 6th and 7th abdominal spiracles, and the perigenital sclerite.

Although recent morphological and molecular studies (McAlpine, 1989, Nirmala et al., 2001, Yeates and Wiegmann, 1999) support the monophyly of the Hippoboscoidea, the phylogenetic relationships among the families, specifically between Nycteribiidae and Streblidae remain unclear. Previous morphological studies placed Streblidae and Nycteribiidae as sister groups supporting the hypothesis of a common origin, and a single lineage evolving with bats (Bequaert, 1954–1957, Hennig, 1969, McAlpine, 1989, Pollock, 1971, Schlein, 1970, Wenzel et al., 1966; Fig. 1A). Hippoboscidae is usually placed as a sister group to the Glossinidae (Fig. 1A), and this group is sister-clade to the bat flies. In the recent molecular analyses of Nirmala et al. (2001) based on 16S rDNA and 18S rDNA, however, Nycteribiidae and Streblidae never formed a monophyletic group suggesting independent origins of their association with bats (Fig. 1B). Also, Glossinidae and Hippoboscidae did not form sister groups. However, the limited number of bat fly species represented by DNA sequences in these analyses (three species) did not allow for a conclusive solution (Nirmala et al., 2001). Thus, no extensive formal analysis has been conducted yet to elucidate evolutionary relationships among the bat fly taxa.

The goals of this work are: (1) to explore the evolutionary relationships between Nycteribiidae and Streblidae, and among their subfamilies and genera, (2) to explore the hypothesis of a single versus multiple independent events of bat fly association to bats in general, and to Micro- and Megachiroptera in particular, and (3) to explore the patterns of contemporary bat fly distribution in light of their phylogeny by means of dispersal-variance analysis. To accomplish these goals, we use a molecular dataset composed of nuclear (18S rDNA) and mitochondrial (16S rDNA, COII, and cytB) genes. Additionally, we comment on evidence supporting a rapid radiation of the hippoboscoid groups.

Section snippets

Taxon sampling

Insect specimens were collected in 96% ethanol and stored at −80 °C. Thirty in-group taxa were included in this analysis representing Hippoboscidae (three species), Glossinidae (one species), and bat flies (26 species). Our sampling distribution covers Old- and New World species of both Nycteribiidae and Streblidae. Exemplars of four out of five subfamilies of Streblidae and two out of three subfamilies of Nycteribiidae are part of this study. The subfamilies Nycterophiliinae (Streblidae) and

Sequences and alignment

The aligned nucleotide sequences resulted in an 18S data partition of 1859 bp, a 16S partition of 512 bp, a CoII partition of 663 bp, and a cytB partition of 373 bp. The total dataset (∼3.5 kb) contained 1007 parsimony informative sites, which in the protein coding genes are largely confined to the third codon position. In the nycteribiid taxa, the stem–loops E19-1 (V4 expansion region), and the stem–loops 34, 35, and V6 expansion region of 18S rDNA show a significant length extension in comparison

Bat fly phylogeny

Overall, although the different reconstruction methods vary in topological relationships of the in-group families to each other, all main bat fly clades were recovered consistently. The most striking result of the present analysis is the clear division of the bat flies into an Old World and a New World clade, which is present under all applied optimality criteria. This places the New World Streblidae apart from all Old World taxa (Nycteribiidae + Old World Streblidae), and renders Streblidae

Concluding remarks

The present study was intended to provide a first molecular phylogeny of the ectoparasitic bat flies. The main conclusions of our analyses are:

  • 1.

    Nycteribiidae are monophyletic.

  • 2.

    Streblidae are paraphyletic.

  • 3.

    No conclusive statement regarding the mono- or paraphyly of the bat flies can be made at this point of the analysis.

  • 4.

    Two major clades, the New World clade (being entirely composed of Streblidae) and the Old World clade (uniting the Old World Streblidae and the Old- and New World Nycteribiidae) are

Acknowledgments

We thank Dr. B. Peterson for assistance identifying some specimens for this study, Liz Price, John Jasper, Krupa Patel, Hashim Bin Man, and Gavin Svenson for collecting assistance. This study was funded by the Karl-Enigk-Foundation for Experimental Parasitology (Hannover, Germany), NSF DEB 9983195 and NSF DEB 0120718.

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