Systematics of the blackfly subgenus Trichodagmia Enderlein (Diptera: Simuliidae: Simulium) in the new world

Systematics of the blackfly subgenus Trichodagmia ENDERLEIN (Diptera: Simuliidae: Simulium) in the New World The systematics of the New World subgenus Trichodagmia has been reassessed by employing an integrated taxonomic approach based upon revisionary taxonomy, phylogenetic (cladistics) analysis, and DNA barcoding. This subgenus included several species of great medical importance, which are all morphologically very similar. The history of the taxonomy and classification of the subgenus Trichodagmia has been put into context with other subgenera within New World Simuliidae, while descriptions and keys to the identification of species in this subgenus are also given. The subgenus Obuchovia is here considered a new junior synonym of Trichodagmia, and all its constituents’ species are now placed in the ALBELLUM species group to represent a Palaearctic element within this subgenus. Three new junior synonymies are here proposed: Simulium chiriquiense FIELD is a synonym of S. ethelae DALMAT n. syn. ; S. biuxinisa COSCARON & IBANEZ-BERNAL is a synonym of S. paynei VARGAS n. syn. ; and S. keenani FIELD is a synonym of S. earlei VARGAS, MARTINEZ PALACIOS &DIAZ NAJERA n. syn. A neotype is designated for S. lahillei (PATERSON & SHANNON) and a lectotype for S. pulverulentum KNAB.1 Simulium falculatum ENDERLEIN is transferred from the TARSATUM species group of to the CANADENSE species group based on the morphology of the female genitalia. Two species, S. rivasi RAMIREZ PEREZ and S. oviedoi RAMIREZ PEREZ, are transferred from the TARSATUM species group to the subgenus Psilopelmia based on the morphology of the male gonostyle and the ventral plate. Keys to separate all species groups and species based on the adults, pupae and larvae are also provided. The phylogeny and classification of the subgenus Trichodagmia is delineated using a cladistic analysis of 63 taxa based on males, females, pupae and larvae, including two species belonging to the subgenus Aspathia and two species of the subgenus Simulium s.str. that served as outgroups. Analysis of the original full data set [67 taxa and 67 characters] with multistate characters treated as unordered under equal weights led to poorly resolved trees, with many polytomies within TARSATUM [= old subgenus Hemicnetha] and CANADENSE [= old subgenus Hearlea]. Nonetheless, the ALBELLUM [= old subgenus Obuchovia] and PICTIPES [= old subgenus Shewellomyia] species groups, and some clades within the CANADENSE species group were well supported. In the most parsimonious cladograms, the position of S. falculatum was problematic as it was placed basal to Trichodagmia. The position of S. jeteri, albeit within the ORBITALE [= old subgenera Trichodagmia + Thyrsopelma of MIRANDA-ESQUIVEL & COSCARON, 2001] clade, was also poorly resolved. This was certainly due to the numerous missing data in these two taxa. Therefore, they were removed from the data set together with other taxa in which three life stages (> 70% of characters) were missing (e.g. S. paracarolinae and S. tarsale). A second analysis was then performed with 63 taxa and 67 characters. In this analysis, the Strict Consensus Tree was better resolved and certain clades within the expanded concept of Trichodagmia (sensu SHELLEY et al., 2010) were recovered as monophyletic with high support values. The ALBELLUM species group is monophyletic in a sister-group relationship with the other species groups in Trichodagmia (sensu SHELLEY et al., 2010). The ORBITALE species group clade was recovered as monophyletic by a unique combination of seven characters with 89% bootstrap support. In this clade, all species close to S. guianense s.l. were better diagnosed by a combination of four characters, one of which (male ventral plate with a globular median process) was unique to this group. The position of S. hirtipupa is better resolved in the latter clade by the presence of black spiniform setae in the frontoclypeus and thorax of the pupa. In contrast, the TARSATUM and CANADENSE species groups were diagnosed by only four and five characters, respectively. Within the CANADENSE species group only species with larvae having sclerotized plates in the posterior region of the abdomen were well resolved. Species in the TARSATUM group were homoplastic. The PICTIPES group is only diagnosed by homoplasies, but the combination of these characters is unique to this clade (polythetic taxon). In general, this study supports some of the taxonomic changes proposed in SHELLEY et al. (2010), in which the subgeneric-names Hearlea, Hemicnetha, Shewellomyia, Trichodagmia + Thyrsopelma (sensu MIRANDA-ESQUIVEL & COSCARON, 502 2001) are treated as species groups within the subgenus Trichodagmia. Moreover, this study also supports the proposal of Obuchovia as a junior synonym within the clade Trichodagmia to represent the ALBELLUM species group. The utility of the COI DNA barcoding methodology for identification of species in the subgenus Trichodagmia and related taxa has been tested. In total, 24 morphospecies within the current expanded morphological concept of Trichodagmia were analyzed. In addition, three species of the subgenus Aspathia and 10 species of the subgenus Simulium s.str. were also included in the analysis because of their putative phylogenetic relationship with Trichodagmia. Within the barcoding neighbour-joining tree, most of the specimens were grouped together according to morpho-taxon (species groups and species). Mean genetic distance amongst groups (morphospecies) averaged 11.2% (ranged 2.8-19.5%), whereas intraspecific genetic divergence within morphologically distinct species averaged 0.5% (range 0-1.3%). In known species complexes, maximum values of genetic divergence (3.28-3.79%) indicate the probable presence of cryptic diversity. DNA barcoding achieved nearly 100% success in identifying all specimens of the subgenus Trichodagmia and related taxa. The existence of well defined groups within S. piperi, S. duodenicornium, S. canadense and S. rostratum highlighted the possible presence of species complexes in these taxa. In addition, the suspected presence of a sibling species in S. paynei and S. tarsatum among populations of Belize, Costa Rica, and the USA is confirmed. The use of shorter barcodes (midi and minibarcodes) from specimens held in collections was problematic with regards to the DNA quality and PCR success. However, in the cases that a readable sequence was obtained, they were sufficient for reliable species identification. With regards to the different extraction and preservation techniques tested, larvae preserved in diluted Carnoy’s (10% acetic acid) provided full DNA barcodes. Furthermore, legs added directly to the PCR mix from freshly collected individuals provided full length barcodes sequences. However, specimens of more than 10 years old did not yield good PCR products. In short, I conclude that DNA barcoding in combination with a morphological benchwork platform is an effective approach for identification and delineation of species in the subgenus Trichodagmia, and the discovery of hidden diversity in this taxon.