Crane flies ( Diptera , Tipuloidea ) from southern Neotropical salt marshes : survey with DNA barcoding

Crane flies are the most diverse group within Diptera, but they are rarely studied in coastal ecosystems. Considering the scarcity of information on the biology and ecology of this group in the Neotropics, and the sparse literature available for taxonomic identification, we developed a descriptive checklist that incorporates morphology and DNA barcoding. We also created a generic identification key for crane flies of southern Brazilian salt marshes. We sampled crane flies continuously at three areas along the Patos Lagoon salt marshes over one year. A total of 14 genera/subgenera, 6 species, and 12 morphotypes belonging to Limoniidae and Tipulidae were identified. Distribution ranges of Symplecta cana (Walker, 1848) and two Ormosia Rondani, 1856 species were expanded. mtDNA COI sequences were compared to the BOLD and NCBI databases, but were matched only at the family level. Therefore, we provided sequences to both platforms, updated to the genus level. We found low (0.00-0.03) intraspecific and high (0.11-0.25) interspecific molecular differences indicating that the mtDNA COI region is adequate for distinguishing species within the Tipuloidea. The Dicranomyia Stephens, 1829 species complex showed low genetic difference, indicating that they could be one species with high morphological plasticity. This study will serve as a basis for future research on insects of Neotropical salt marshes.

The superfamily Tipuloidea is the richest within the order Diptera.It is subdivided into Cylindrotomidae, Limoniidae, Pediciidae, and Tipulidae sensu stricto.Approximately 3,500 crane fly species are recognized throughout the Neotropical region, of which around 99.6% are in the families Limoniidae and Tipulidae (De Jong et al., 2008;Oosterbroek, 2018).Crane flies are non-hematophagous flies that are extremely important in the trophic webs they participate in, generally exhibiting a detritivorous diet during their larval phase.Adults generally do not feed, but some will take nectar, and perhaps pollen, and water.The large corporal mass of crane flies, along with their occupation of transitional ecosystems (e.g., salt marshes), make these insects a potential and important trophic link between aquatic and terrestrial environments (e.g., Baxter et al., 2005).This group is widely distributed over almost all environments, and includes species that occupy wetlands such as salt marshes (Rogers, 1932;Autio et al., 2013).
Salt marshes are typical coastal habitats of mid-and high-latitude areas (Stevens et al., 2006), including the southern Neotropical region.They are characterized as transition areas between estuaries and land, display few but numerous plant species, and are dominated by a fauna and flora that tolerate rapid variations in salinity (Costa & Marangoni, 2010).At Patos Lagoon Estuary, southern Brazil, estuarine flow and precipitation levels tend to increase salinities at these salt marshes during the summer, and decrease them in the winter (D'Incao et al., 1992).The margins of this estuarine environment are dominated by halophyte plant species such as Spartina alterniflora and S. densiflora (Poaceae), as well as Myrsine parvifolia (Primulaceae) shrubs (Costa et al., 1997).
Other than their original descriptions, few studies have reported and updated the occurrence of crane fly species across the Neotropical region (see Ribeiro et al., 2007;Ribeiro & Santos, 2016).Additionally, research at salt marshes is commonly focused on species such as fish (e.g.Contente et al., 2010;Campos et al., 2015), polychaetes, crustaceans (e.g.Mackenzie et al., 2015) and birds (e.g.Britto & Bugoni, 2015), but there are rare studies on insect groups conducted at these environments (see Giberson et al., 2001;Mackenzie, 2005;Dummel et al., 2011;Bolico et al., 2012;Gantes et al., 2013;Rodrigues et al., 2017).In order to support studies on the classification, biogeography, and ecology of these groups, it is necessary to identify the insect species that inhabit these environments, preferably with high taxonomic resolution, as suggested by Lenat & Resh (2001).Morphological and DNA barcoding methods are frequently combined for taxonomic determination, the latter based mainly on mitochondrial DNA Cytochrome Oxidase Subunit I (COI) sequences for specific identification of animals (Hebert et al., 2004).Therefore, we conducted morphological identification, provided species-specific COI barcode sequences, and developed an identification key for the Tipuloidea genera that occur at salt marshes of the southern Neotropical Region.

MATERIAL AND METHODS
Study area and sampling.The salt marshes of the Patos Lagoon Estuary are one of the largest in Brazil, covering over 70 km² (Costa et al., 1997).We sampled three salt marsh areas: upper estuary (31°53'33"S; 52°14'33"W), middle estuary (32°02'01''S; 52°10'45"W), and lower estuary (32°10'65"S; 52°08'52"W) (Figure 1).Two Malaise traps with ethanol-filled collection cups were set up at each sampling area, close to the dominant vegetation, for a total of six traps.Malaise traps are flight intercept traps known for efficacy in capturing winged insects, including crane flies.Sampling was conducted continuously over twelve months, from August 2015 to July 2016.Collection cups were changed every 15 days, and collected insects were removed for identification and preservation in 70% ethanol.Specimens were deposited at the Fundação Zoobotânica do Rio Grande do Sul -FZB/ RS.Sampling was conducted under the Brazilian system for biological sampling (SISBIO) license number 50253-1.
Species distribution.Large scale species distributions are from the Catalogue of the craneflies of the World (Oosterbroek, 2018).However, this catalogue does not differentiate between different regions within Brazil, which is important for such a large country with pronounced regional variations.

RESULTS
We collected a total of 5,248 specimens distributed over 18 crane fly species/morphospecies at the salt marshes of Patos Lagoon Estuary.Three species belonged to the family Tipulidae and the other 15 to Limoniidae.This included members of a Dicranomyia species complex.We also found one taxon that we could not identify to genus.Symplecta pilipes (Fabricius, 1787) and Rhipidia domestica amazonensis Osten Sacken, 1860 represented 68% of all crane flies collected.A total of 43 sequences, corresponding to the 18 species, were compared with those in BOLD.Thirty of these presented ≥ 97% similarity compared to the existent sequences in the database, but none had specific identification.The majority of these matched sequences are from Argentina, but matches included sequences of Nephrotoma sp. from Ecuador and Geranomyia from the U.S.A., Dominican Republic, and Haiti.All haplotypes of the Dicranomyia complex (~91.43%),Gonomyia (Neolipophleps) sp.(~91.37%),Polymera inornata Alexander, 1913 (~88.53%),P. obscura Macquart, 1838 (88.11%), and Symplecta cana (95.05%) showed the lowest similarities with BOLD sequences, and were therefore considered new.We observed overall high interspecific COI differences among Tipuloidea species (between 0.11 and 0.25) and low intraspecific differences (between 0.00 and 0.03) (Figure 2).Gonomyia (Neolipophleps) sp. was the exception, with intraspecific variation ranging from 0.03 to 0.12.A checklist of taxa listing their authors, references, and FZB/NCBI deposit numbers is shown in Table 1.It was hard, and in some cases impossible, to identify at specieslevel because C. P. Alexander, whom mostly described them, used mainly coloration or other non-usual taxonomic characters.Geranomyia, Ormosia and the unidentified genus were not included.Three listed species -Ozodicera sp., and the two morphotypes of Toxorhina -did not have enough specimens for molecular analysis.An identification key (modified after Gelhaus, 2009) and list of genera/species description is presented below.Flagellomeres of males and females oval to elongated shape (Fig. 16) ....……………………………….Dicranomyia 14.A 2 "S" shaped distally; cell r 3 with crossvein (Fig. 17 Diagnosis.Morphology of antennae usually simple, antennae with elongated flagellomeres (Fig. 16).Wings, if well developed, with relatively short Sc vein, usually ending approximate to the base of Rs or before mid-length of Rs, never reaching the fork of Rs.

Geranomyia Haliday, 1833
Diagnosis.Its members are characterized by both having a supernumerary crossvein at about midlength of wing cell Sc and the elongate mouthparts of the adult fly (Fig. 9).

Gonomyia (Paralipophleps) Alexander, 1947
Diagnosis.Two branches of Rs strongly divergent, at least at wing margin.Cell dm present.Wing with conspicuous dark-brown pterostigmal spot.

Polymera Wiedemann, 1821
Diagnosis.Males with extremely elongated antennae, at least as long as body, and usually clothed with long, delicate, outstretched hairs.Antennal segments elongatecylindrical or bi-nodose.

Polymera obscura Macquart, 1838
Diagnosis.Antennal segments bi-nodose, darker on the nodes, lighter at the constriction.Wings gray with indistinct rounded clouds at the origin of Rs.

Polymera inornata Alexander, 1913
Diagnosis.Antennal segments bi-nodose, lighter at the base and apex of each segment, but not producing an annulated effect as in P. obscura.Differs from other species in the extreme recession of the cross-vein r (i.e., r equidistant between tip of Sc2 and tip of R 1 ).
Previous geographical distribution: Guyana.

Rhipidia domestica amazonensis Osten Sacken, 1860
Diagnosis.Mesotonal praescutum with dark longitudinal lines with wing markings large, scanty, confined to the neighborhood of veins.Wings tinged with brown in R. domestica amazonensis compared to the hyaline wings of R. domestica domestica.
Previous geographical distribution: East Brazil Symplecta (Symplecta) Meigen, 1830 Diagnosis.Wings usually with spots and seams on cross-veins, base of Rs, tips of Sc and R 1 , and middle and tip pf A 2 .Supernumerary cross-vein in cell R 3 .A 2 more or less sinuous (Fig. 17).

Symplecta (Trimicra)
Diagnosis.Subgenera Trimicra is distinguished by the antennae, in which the apical three segments are abruptly slenderer than the preceding ones.

Symplecta (Trimicra) pilipes (Fabricius, 1787)
Diagnosis.On male, vein Rs with dense row of long hairs.Mainly larger size, unpatterned wings and hairy legs distinguish this species from others in Trimicra.
Previous geographical distribution.The only cosmopolitan species crane fly (Andrew, 2000).

Zelandotipula Alexander, 1922
Diagnosis.Wing with cell r 3 constricted at midlength.Wing often with spot or cloud over origin of Rs, and at base of middle of cell bm.
Zelandotipula neurotrichia (Alexander, 1962) Diagnosis.Wings gray, conspicuously patterned with brown, including cord and adjacent veins.Veins beyond cord with conspicuous macrotrichia, including all veins from R 2+3 to distal section of vein Cu 1 .Abdomen with segments conspicuously bicolored, disk yellow and with brown margins.
Previous geographical distribution: Bolivia.

DISCUSSION
The richness of insects in salt marsh environments is commonly underestimated because of the general assumption that this environment is hostile for insects with aquatic/semiaquatic larval phases.However, previous studies have shown that relatively high salinity does not hinder the presence of Diptera species in these habitats (Williams & Williams, 1998;Giberson et al., 2001;Mackenzie, 2005;Silberbush et al., 2005;Boix et al., 2008).This is supported by our results, particularly for the species richness in Limoniidae.
Some of the taxa we identified are noteworthy: Dicranomyia is the largest genus in the Limoniidae, Iheringia, Série Zoologia, 109: e2019013 representing around 10% of Neotropical species (Oosterbroek, 2018).Representatives often display significant morphological variability, confusing species distinctions, so it is common for these to be treated as species complexes (Nitta & O'Grady, 2008;Goodman & O'Grady, 2013;Salmela et al., 2014;Starý & Stubbs, 2015).Species within our Dicranomyia complex were all genetically similar, which suggests that their different morphologies, especially in terms of wing venation, are a morphological plasticity of the group.This is the first report of the genus Ormosia for the Neotropical region, and consequently for Brazil.We expanded the ranges for Polymera inornata and Symplecta cana, with the latter now for both continents of the New World (previously only reported in North America).Symplecta pilipes is a cosmopolitan species, and combined with the American Rhipidia domestica amazonensis, these two species exhibit high densities in Neotropical salt marshes (see also Rodrigues et al., 2017).
De Jong et al. (2008) argue that all Tipuloidea species/ subspecies are restricted to determined biogeographic regions, with only a few sharing neighboring regions.Most mtDNA COI sequences found in this work were highly similar to those from Argentina, Costa Rica, Dominican Republic, Ecuador, and Nicaragua (Neotropical region), and different from those from regions such as Canada and U.S.A. (Nearctic region), which corroborates a relatively limited range distribution of these crane fly species, and reinforces the endemic characteristic of this group in the Neotropics.Floyd et al. (2009) alert that using COI sequences for identification of insect species can lead to overestimation of richness.However, we found low intraspecific (< 0.03) and high interspecific molecular differences (0.11-0.25) among the sampled insects, indicating that the mtDNA COI region is a useful tool for species distinction in the Tipuloidea.This has also been observed by Hebert et al. (2004).This molecular marker has been shown to be useful for the identification of other Diptera species with non-conspicuous morphological diagnostic characteristics, such as blackflies (Diptera: Simuliidae; Pramual et al., 2016), mosquitoes (Diptera: Culicidae; Kumar et al., 2007), and sandflies (Diptera: Psychodidae; Nzelu et al., 2015).Gonomyia (Neolipophleps) sp. was the only species with high intraspecific distance (0.03-0.12), because of one or more divergent specimens.This differentiation between specimens was not detected morphologically by wing venation and male genitalia, but could indicate the existence of another Gonomyia (Neolipophleps) species.
The current literature on this superfamily at the Neotropical region is sparse and fragmented, consisting primarily of the original descriptions of C. P. Alexander.In this manner, it becomes necessary a complete review for regional Tipuloidea species.The insertion of new and generically-identified sequences in NCBI is an important step that will provide support for researches using molecular tools for identification and/or phylogenetic studies of Tipuloidea.Additionally, mtDNA COI barcoding is a useful tool for Tipuloidea species identification.This is one of the first works to list and barcode Tipuloidea species from Neotropical salt marshes, decreasing the large gap in information on crane flies at the region, and aiding in future studies aimed towards exploring the life cycles and/or ecology of these insects.

Fig. 1 .
Fig. 1.Sampling areas at salt marshes of the Patos Lagoon Estuary, Rio Grande do Sul, southern Brazil.

Fig. 2 .
Fig. 2. DNA barcoding gap analysis, with frequency of intra and interspecific distances in COI sequences among Tipuloidea species.