Taxonomic review and phylogenetic analysis of fifteen North American Entomobrya (Collembola, Entomobryidae), including four new species

Abstract The chaetotaxy of 15 species of eastern North American Entomobrya is redescribed in order to determine potential characters for the diagnosis of cryptic lineages and evaluate the diagnostic and phylogenetic utility of chaetotaxy. As a result, four new species (Entomobrya citrensis Katz & Soto-Adames, sp. n., Entomobrya jubata Katz & Soto-Adames, sp. n., Entomobrya neotenica Katz & Soto-Adames, sp. n. and Entomobrya unifasciata Katz & Soto-Adames, sp. n.) are described, and new diagnoses are provided for Entomobrya assuta Folsom, Entomobrya atrocincta Schött, Entomobrya decemfasciata (Packard), Entomobrya ligata Folsom, Entomobrya multifasciata (Tullberg), and Entomobrya quadrilineata (Bueker). Furthermore, previously undocumented levels of intraspecific variation in macrosetal pattern are reported, tempering the exclusive use of chaetotaxy for species delimitation. Phylogenetic relationships, estimated using both morphological and molecular data, indicate that Entomobrya is likely paraphyletic. The phylogenies also suggest that unreliable character homology, likely fostered by Entomobrya’s profusion of macrosetae, may limit the phylogenetic utility of chaetotaxy in groups characterized by an abundance of dorsal macrosetae.


Introduction
Studies concerning species delimitation and taxonomy of Collembola have traditionally relied on comparative morphology. For some groups, however, uncertain homology, intraspecific variation, and characters of difficult observation have limited the utility of morphological characters as tools for species diagnosis and phylogenetic inference. Recent advances in DNA sequencing technology have provided scientists with additional ways to delimit groups that lack informative morphology, such as cryptic species complexes (Frati et al. 1994(Frati et al. , 2000Carapelli et al. 1995Carapelli et al. , 2005Simonsen et al. 1999;Soto-Adames 2002;Burkhardt and Filser 2005;Cicconardi et al. 2010Cicconardi et al. , 2013Felderhoff et al. 2010;Porco et al. 2012;Zhang et al. 2014c;Katz et al. 2015), but in practice most species diagnosis remains dependent on fixed characters, only obtained by rigorous morphological examination (Will and Rubinoff 2004).
Taxonomy and species delimitation of the genus Entomobrya Rondani, 1861 has been especially problematic due to intraspecific morphological variation and a general lack of informative taxonomic characters (Christiansen 1958b;South 1961). Conspicuous dorsal color patterns exhibited by members of this genus originally served as a practical means for species diagnosis (Bonet 1934). Other characters such as claw structure, antennal sense organs, setae types, and chaetotaxy of the male genital plate were introduced in Christiansen's (1958b) revision of North American Entomobrya, who attributed many color forms to intraspecific variants. These new characters marginalized color pattern as an exclusive diagnostic tool, but some are difficult to observe. Szeptycki's (1979) publication on the phylogenetic significance of dorsal chaetotaxy, introduced standard nomenclature for dorsal setae, providing a practical system to assess element homology between species. This system established chaetotaxy as the most important tool for species diagnosis in the family Entomobryidae and in theory, provided many additional characters to infer phylogenetic relationships (Soto-Adames 2008). Bellinger (1980, 1998) later combined traditional morphology with chaetotaxy in order to clarify species boundaries. Jordana and Baquero (2005) then simplified Szeptycki's (1979) system of body chaetotaxy and incorporated additional nomenclature for elements on the head later formalized by Soto-Adames (2008). Following this new nomenclature system, Jordana (2012) was able to delimit approximately 270 Palearctic species in subfamily Capbryinae and tribe Entomobryini using chaetotaxy.
Chaetotaxy has surely proven to be a valuable tool for springtail taxonomists, but not without complications. For some groups, especially those characterized by large numbers of setae, such as Entomobrya, the homology of macrosetae is not always clear. Intraspecific variation, apparent differences in setae arrangements, and differences of setae types make it difficult to determine homology between species (Soto-Adames 2008). Despite these complications, chaetotaxy still provides fixed observable differences that have been widely applied to successfully delimit species boundaries (Christiansen and Bellinger 1998;Carapelli et al. 2001;Jordana and Baquero 2005;Soto-Adames 2010;Jordana 2012). However, its utility as diagnostic and phylogenetic characters is clearly dependent on a detailed assessment of intraspecific variation and homology between species of interest. Christiansen and Bellinger's (1998) "The Collembola of North America" is the current authority for Nearctic Collembola species identification. However, some of their descriptions of Entomobrya express the need for re-examination due to a lack of informative characters for clear delineation of species boundaries, and high levels of observed intraspecific chaetotaxy and color pattern variation that suggest the presence of cryptic species complexes. In addition, their descriptions of chaetotaxy are limited and often unclear. Therefore, the primary goal of this study is to examine and document Entomobrya chaetotaxy to provide detailed descriptions and figures in order to clarify species boundaries and to simplify the diagnosis of eastern North American Entomobrya species. A total of 15 species of North American Entomobrya that occur east of the Mississippi River are examined and described with special emphasis on chaetotaxy and color pattern, including three cryptic species lineages identified by Katz et al. (2015) and two new species. Additionally, phylogenies, incorporating both the morphology described in this study and molecular COI sequences from Katz et al. (2015), are presented in order to explore how chaetotaxy and other morphological characters affect phylogenetic estimation.

Specimen collection and preparation
Approximately 146 specimens, representing 15 Entomobrya species (11 previously reported, 4 new), were examined in detail throughout the course of this study. Historical collections of Entomobrya from the Illinois Natural History Survey were also examined, but were not useful for the present study. Specimens preserved in 70% EtOH are old (e.g., collected prior to 1980) and in poor condition; although color pattern is often preserved, chaetotaxy and other small, but diagnostic, characters are extremely difficult to observe with confidence. Therefore, more recent material was needed to study details of chaetotaxy and other characters. Most specimens examined were collected by the senior author between 2011 and 2012 or otherwise provided by colleagues from localities throughout the USA, east of the Mississippi River. In the Material Examined sections it is assumed that the senior author collected all material, unless otherwise noted. Specimens were usually collected from leaf litter and extracted using a Berlese funnel or hand collected from bark and vegetation with an aspirator. Table 1 lists all Entomobrya species reported from North American.
Individuals sampled were sorted under a dissecting microscope to morphospecies according to color pattern and photographed to record dorsal thoracic and abdominal color patterns prior to slide mounting. All specimens were cleared with Nesbitt's solution and mounted on Hoyer's medium (Mari Mutt 1979) in preparation for light microscopy. The heads of specimens generally take longer to clear so they were dissected and mounted Table 1. List of all Entomobrya species reported from North America. Of the 31 species, 15 were examined for this study and 16 were not included.
Labial appendage: Christiansen (1958b) incorporated the ratio of labial appendage on papilla E ( Fig. 2A) to papilla length, even after recognizing the potential limitations of relative ratios (Christiansen 1954). The deformation of these soft body parts after slide mounting and pronounced variation between instars makes these measurements unreliable and not practical for diagnostic purposes (South 1961).
Labial chaetotaxy: Labial palp proximal setae, labial triangle setae, and post-labial setae have been shown to be taxonomically informative for other genera Christiansen 1993, 1997;Soto-Adames 2010), but generally lack any useful variation in North American Entomobrya. For this study, all descriptions of labial chaetotaxy follow nomenclature developed by Chen and Christiansen (1993).
Dorsal chaetotaxy: The introduction of "stable" character systems for dorsal macrosetae (Szeptycki 1979;Jordana and Baquero 2005;Soto-Adames 2008) have provided a large set of characters that are relatively easy to observe and compare among different  species. Entomobrya are extremely setaceous (polychaetotic), providing many diagnostic characters. However, the abundance of macrosetae and observed variation among instars (including among adult instars) makes homology assessment of each element difficult. The macrosetae inserted external to the sensilla on abdominal segments (Abd.) 1-3 and external to the lateral bothriotricha on Abd. 4 were not included in descriptions or analysis due to extensive variation and difficulty of observation. The dorsal chaetotaxy of the head and along the dorsal median line on the mesothorax (Th. 2), metathorax (Th. 3), Abd. 1-3 and macrosetae internal to the lateral bothriotricha on Abd. 4 are easy to observe and relatively stable, thus are emphasized in the descriptions and analysis. The chaetotaxy of Abd. 5 lacks useful variation in specimens observed in this study. Thoracic zones (Fig. 3) originally described by Szeptycki (1979), differ in number of macrosetae among Entomobrya species and may provide some initial direction in the identification process.
Trochanteral organ: Differences in number and arrangement of small, spine-like setae on the trochanter, termed the trochanteral organ, have also been used to support the identification of some species (Christiansen 1958b). These setae are not only difficult to observe, but intraspecific variation limits their use for Entomobrya species delimitation (South 1961). However, the setal pattern, rather than the presences/absence or specific seta, seems to separate some species.
Male genital plate: Differences in chaetotaxy of the male genital plate can accurately delineate many North American Entomobrya species (Christiansen 1958a(Christiansen , 1958b. However, males with a well-developed plate are uncommon in the samples examined and when present, plates are difficult to observe under light microscopy, requiring electron microscopy in order to easily discern characters with certainty (Fig. 4).
Mucro and tarsal claw: Ratios of relative positions of mucronal and ungual teeth do not usually deform by the mounting process, but they depend heavily on the angle or position of the slide mount in order to properly standardize relative measurements between individuals. Additionally, these characters present few discernable differences between Entomobrya species and have been noted to be of little taxonomic value for European Entomobrya (South 1961). There are marked differences in the distance between dorsal tooth and lateral teeth on the unguis between Entomobrya species. However, these measurements are difficult to quantify due to variation and mounting inconsistencies.
Anatomical measurement ratios: Some authors have used relative anatomical length or distance ratios for Entomobrya species separation (Christiansen and Bellinger 1998;Jordana 2012). Although measurement ratios may provide some level of diagnostic utility, high levels of variation in ratios (Christiansen 1954(Christiansen , 1958b, deformation of soft tissues, and variable final position of mounted specimens reduce character consistency and reliability, thus ratio measurements are not used here as diagnostic characters.

Character nomenclature, abbreviations, and symbols
Descriptions of adult dorsal macrosetae provided in this study follow the dorsal trunk chaetotaxy nomenclature established by Szeptycki (1979) and the dorsal head chaetotaxy from Jordana and Baquero (2005) and Soto-Adames (2008). References to thoracic and abdominal segments are abbreviated as Th. (2-3) and Abd. (1-6) respectively. Symbols used in chaetotaxy descriptions are presented in Figure 5. There are seven general morphological structures, including three types of setae, recognized for the purpose of this study. Macrosetae are the primary setae, characterized by a large socket, long shaft, and are usually apically truncate or blunt (Fig. 6A). Microsetae are common type 5 (Christiansen 1958b), ciliate, short, thin, acuminate setae with very small sockets (Fig. 6A). Mesosetae are morphologically similar to microsetae, but are ostensibly longer, with larger sockets (Fig. 6A). They tend to be smaller and thinner than macrosetae, but substantial variation in length and size of both setae types cause overlap. Bothriotricha (Fig. 6A) are specialized setae characterized by unique morphology; long, thin, with conspicuous ciliation. There are also two types of short, smooth, acuminate, spine-like sensilla: type 1 (S-chaeta of Zhang and Deharveng 2015) is long and more common and occur on all thoracic and abdominal segments; while type 2 (Smicrochaeta of Zhang and Deharveng 2015) is short, sometimes slightly blunted, usually paired with type 1, and is only present on Th. 2, Abd. 1, and Abd. 3. Pseudopores are relatively difficult to observe and resemble sockets of macroseta, but are generally shallower and lack a thickened socket wall (Fig. 6B).

Phylogenetic analysis
In order to investigate the effects chaetotaxy and other morphological characters have on phylogenetic relationships, Bayesian and maximum likelihood phylogenetic analyses were conducted using MrBayes v. (Folsom) was selected as the outgroup. Character state assignments (Suppl. material 1) were attained through observation of material collected for this study, except for P. violenta and Seira dowlingi (Wray), which were obtained from Soto-Adames (2010) and Soto-Adames (2008) respectively. Morphological characters were analyzed under the Mk model of evolution (Lewis 2001), with rate variation among characters (gamma distribution), four independent runs, starting from random trees, four Markov chains (temp=.5), and 25,000,000 generations, sampling every 1000 th generation. Default values were used for all other parameters.
The combined analysis incorporating complete COI sequences (1539 bp) for 89 exemplars (See Katz et al. 2015 for specific details regarding specimens, Gen-Bank accession numbers, gene choice, DNA extraction, amplification, sequencing, and primer development) with P. violenta as the outgroup. The appropriate model of sequence evolution (GTR+I+G), was selected using jModeltest (Posada 2008), whereas the Mk model (Lewis 2001) was implemented for the morphology dataset. Morphology was not examined for sequenced individuals due to the destructive process of DNA extraction; therefore character states were attained from other individuals of the same species or from descriptions in the literature (Soto-Adames 2008, 2010 and were added to corresponding OTU's in the matrix. Bayesian analyses were conducted using MrBayes on the CIPRES Science Gateway (Miller et al. 2010), convergence was assessed by observation of average standard deviation of split frequencies values below p < 0.003, a 25% burn-in was used, and all posterior probabilities and consensus trees were computed in MrBayes. The maximum likelihood analyses were conducted using RAxML's rapid bootstrap algorithm incorporating 1000 bootstrap replicates.  (1), mesoseta (2), bothriotricha (3), the dotted line represents the medial division of Abd. 3 B comparison between a pseudopore (1) and macroseta socket (2). Scale bars = 20 µm.

Character state assignments
Dorsal setae were identified by their relative positions to bothriotricha, sensilla, pseudopores, and to neighboring setae, following descriptions provided by Szeptycki (1979), Jordana and Baquero (2005), and Soto-Adames (2008). All macrosetae labeled in figures for species descriptions were included as characters in the phylogenic analysis (See Suppl. material 1 for morphological character matrix). Macrosetae external to sensillum on Abd. 1-3 and external to bothriotricha on Abd. 4 were not included in descriptions or analysis due to uncertain homology. Characters states for setae were defined as present, absent, or polymorphic (i.e., variable between individuals of the same species). Macrosetae were considered absent if meso-or microsetae were present in corresponding position. Polymorphic states are indicated in the figures as dotted circles. Other morphological characters included in the phylogenetic analysis are number of eye patch setae, labral setae (smooth or ciliate), labral papillae (smooth, single projection, or multiple projections), setae within labial triangle (ciliate or smooth), antennal bulb (absent or present), scales (absent or present), and dental spines (absent or present). See Suppl. material 1 for a complete list of all 179 morphological characters and character states circumscription used in this analysis. This genus characterized by having 8+8 eyes within black or dark blue patches of pigment, a bidentate mucro with a smooth basal spine, basic chaetotaxy formed by type 5 microsetae and the absence of antennal sub-segmentation, scales, dental spines, and differentiated "smooth" setae on the inner surface of the hind tibiotarsus.

Species descriptions and taxonomy
In addition, all species treated here have an apical antennal bulb; main sensilla on 3 rd antennal segment sense organ thin, smooth, blunt and peg-like; differentiated smooth setae on ventral side of 1 st antennal segment of two types, short and spine-like and long and seta-like; labral setae 5,5,4 and smooth; outer maxillary lobe of maxilla with subapical and apical setae smooth and subequal, and sublobal plate with three smooth seta-like appendages; lateral appendage of labial papilla E slightly curved, relatively thick, blunt; all post labial setae type 5; unguis with one outer, two lateral and four inner teeth, and a lanceolate unguiculus; and mucronal spine smooth; and most have all posterior setae of labial triangle ciliate, as M1, r, E, L1, L2, with r significantly smaller than other setae and A1-A5 smooth.   More general morphological descriptions of this genus are provided in Christiansen (1958b), Stach (1963), Bellinger (1998), andJordana (2012). Chaetotaxy provided in the following descriptions follows the nomenclatural systems established by Szeptycki (1979) and Jordana and Baquero (2005) with some modifications.
Informative diagnostic adult chaetotaxy characters of all Entomobrya species treated here are listed in Table 2. These characters were specifically chosen for their ease of observation, stability (lack of polymorphisms), and confident homology. citrensis sp. n. was at first considered to be an undescribed color form of E. assuta, but molecular data provides evidence for their separation (Katz et al. 2015).
Subsequent comparative morphological observations between the two forms show that head macroseta Ps 3 and Abd. 2 macroseta a 3 are both absent in E. assuta, but present in E. citrensis sp. n.; additionally, E. citrensis sp. n. has a complete, dark transverse band located medially across Abd. 4, whereas this band is absent in E. assuta. Labral morphology also separates these species, E. assuta has relatively uniform labral papillae, each with two to three seta or spine-like projection, whereas E. citrensis sp. n. has up to five minute bumps or serrations on the two internal papillae and only two larger spinelike projections on the two external papillae (Fig. 2). It should be pointed out that the last character may be variable and should be used in combination with chaetotaxy and color pattern for diagnosis. Christiansen and Bellinger (1998) reported seven different color forms of E. assuta, each occurring in separate localities across North America. Many of these color forms were not sampled for this study, and in view of the discovery of E. citrensis sp. n. it is possible that some of them may represent distinct species. Future determinations of E. assuta will have to be based on analysis of chaetotaxy and other morphological characters outlined in the present description and not just color pattern. Entomobrya assuta appears to be an intrusion of a southern subtropical or tropical Entomobrya lineage into the Nearctic region. Christiansen and Bellinger (1998) noted that the dorsal and genital chaetotaxy of E. assuta is more similar to tropical rather than Nearctic species. In fact, the reduction in chaetotaxy approaches that seen in E. longiseta Soto-Adames and E. linda Soto-Adames from the Caribbean, more than other Nearctic forms.   3B, 4, 6, 9, 10, 39 Description. Body shape and color pattern. Sexually dimorphic in color pattern and body shape. Males and females with variable but characteristically different color patterns ( Fig. 9). Male body relatively cylindrical, slender, with bright orange background with black pigment usually forming a thick and complete transverse dorsal band covering posterior margin of Th. 2 and all of Th. 3 and Abd. 1, band sometimes absent. Male light form without dark pigment on Th. 2 through Abd. 6 (except sometimes along anterior margin of Th. 2). Male dark form with band covering Th. 2 through Abd. 2, irregular pigment patterns sometimes forming 1+1 orange spots on Abd. 3, and two narrow longitudinal stripes connected by transverse band on posterior margin of Abd. 4 ( Fig. 9A-D). Females with slightly dorso-ventrally flattened body and slightly larger than males. Female color pattern strikingly different from males, white or light yellow background with black, dark blue or purple pigment forming transverse bands across the posterior margins of Th. 2 through Abd. 4. All females with two longitudinal stripes or triangular extensions connected by two transverse bands on Abd. 4; one incomplete medial band and another complete band on posterior margin of Abd 5 ( Fig. 9E-I). Mesonotum white in both sexes, lacking pigment except for a small irregular band across anterior margin. Medial area of Th. 2 relatively transparent and fat bodies visible through cuticle under a dissecting microscope. Both males and females with purple pigment usually extending from apical end of 2 nd antennal segment through apex of 4 th antennal segment.
Head. Apical bulb of 4 th antennal segment located in deep pit, usually simple, sometimes with up to four distinct lobes. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈2.5x short setae. Prelabral setae ciliate. Ornamentation of distal margin of labral papillae with 3-4 small seta or spine-like projections (Fig. 2). Dorsal head chaetotaxy variable (Fig. 10A); macrosetae An' 0 , An 3a2 , An 3a3 , S' 0 , S 6 , Ps 3 , Pi 1 , Pa 3 , and Pm 1i always absent; S 0 usually present, M 3i usually absent, A 6 present in roughly half of observed specimens. Eyes G and H small and subequal; eye patch with 5 setae.
Legs. Trochanteral organ with triangular setal pattern and up to 30 setae. Unguis with 4 internal teeth; basal teeth located approximately middle of inner claw length.
Remarks. Entomobrya atrocincta can be distinguished by the male or female color patterns as described above combined with the presence of macrosetae head S 4i and Abd. 3 a 1 and a 2 and the absence of macrosetae head ps 3 and Th. 2 m 5 (see Table 2 for additional diagnostic characters). The color pattern of female E. atrocincta is virtually indistinguishable from that of E. multifasciata and very similar to E. intermedia, and E. nivalis. Though these species may exhibit slight differences in color pattern, it is easier to differentiate them with the characters outlined in Table 3. Female E. multifasciata and E. atrocincta can be separated by the presence of head macroseta S 4i in E. atrocincta and its absence in E. multifasciata, and morphology of the labral papillae; E. multifasciata has 2-3 large seta or spine-like projections per papillae, whereas E. atrocincta has 3-4 small, seta or spine-like projections per papillae (Fig. 2).
The male form always has a unique orange color, but the distribution of purple pattern is variable. Christian and Bellinger (1998) report four discrete color forms. An additional male color form was collected during this study. The different color forms can commonly be found together within the same population. There are no significant morphological or COI DNA sequence differences between male forms and variation in color pattern (male and female) is attributed to intraspecific variation (Katz et al. 2015). The most common male form collected in this study ( Fig. 9A) is somewhat similar to E. clitellaria. However, the two forms can be easily separated by chaetotaxy (Table 3).
Sexual dimorphism in this species has caused serious taxonomic confusion due to the similarity of female pattern to E. multifasciata and E. nivalis. Ramel et al. (2008) first described the sexual dimorphism of E. atrocincta from Greece and even noted that records of E. multifasciata may be misidentified E. atrocincta females. However, Jordana (2012) separated most European specimens and classified them as E. nigrocincta Denis based on chaetotaxy, synonymizing Ramel et al.'s (2008) descriptions with E. nigrocincta. Jordana (2012) attributes the sexual dimorphism (the same displayed by the specimens collected in North America and included in this study) to E. nigrocincta only, keeping the description by Christiansen (1958b) valid for E. atrocincta. Molecular data confirm that different color forms represent different sexes of the same species (Katz et al. 2015), demonstrating the presence of sexual dimorphism in North American E. atrocincta. However, species diagnosis remains unclear. Chaetotaxy outlined for both E. atrocincta and E. nigrocincta by Ramel et al. (2008) and Jordana (2012) do not match the specimens examined in this study (Table 4). The excessive intraspecific variation in chaetotaxy observed in these specimens raises concern about basing species diagnosis strictly on discrete chaetotaxic characters. Further molecular analysis of European populations is needed in order to elucidate the correct taxonomic status and distribution of these two species.
The original description of E. atrocincta by Schӧtt (1896) was based on the male form collected from California. Christiansen's (1958b) descriptions and methods for species delimitation placed heavy emphasis on the male genital plate, which may have led to his inadvertent omission of the female form. Given that the combination E. atrocincta Schӧtt, 1896 has priority over E. nigrocincta Denis, 1923, and also because the holotype designated for E. atrocincta was collected in North America, the specimens along with the descriptions outlined in this study have been assigned to E. atrocincta.
Distribution. North America, Hawaii and possibly Europe. Records of E. multifasciata in North America and Hawaii (Christiansen and Bellinger 1992) are suspect due to their similarity to the female E. atrocincta color form. A considerable number of collections of E. atrocincta from Hawaii also include E. multifasciata, indicating the species is also sexually dimorphic in the Pacific Islands (Christiansen and Bellinger 1992).    (2012), Ramel et al. (2008), and Christiansen and Bellinger (1998).
Characters represent the number of dorsal macrosetae present in fields (H1-A10) outlined by Jordana and Baquero (2005) and Jordana (2012). Clear differences between descriptions are highlighted in bold and underlined. Parentheses indicate a rarely observed state. Question marks indicate the characters were not included in the description.  (2012) and Ramel et al. (2008). 3 Description of E. atrocincta provided by Jordana (2012). 4 Description of E. atrocincta provided by Christiansen and Bellinger (1998).    antennae lightly colored with brown or purple pigment, usually with a white area on distal half of 1 st antennal segment. Juvenile pattern distinct from adult, yellow background without dark pigment except for eye patch; legs, furcula, Abd. 6 pale, lacking all dark pigmentation; antennae with light purple pigment. (Fig. 11B) Head. Apical bulb of 4 th antennal segment usually bilobed, rarely simple. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈ 3-4× short seta. Prelabral setae ciliate. Ornamentation of the distal margin of the labral papillae with a single seta or spine-like projection (Fig. 2). Labial papilla E with lateral appendage reaching just above tip of papilla. Labial triangle in one individual with 2 small supplementary ciliate microsetae internal to M1. Dorsal head chaetotaxy as in Figure 12A: macrosetae An' 0 , A 6 , S' 0 , S 6 , Ps 3 , Ps 5 , Pi 1 , Pi 3 , and Pm 1i always absent; An 3a3 usually absent (present on one side in one specimen). Eyes G and H small and subequal. Eye patch with 5 setae.
Thorax. Thoracic chaetotaxy abundantly developed, highly variable with many supplemental macrosetae. Th. 2 macrosetae p 6e and p 6ep absent and macrosetae m 4i2 , m 4i3 polymorphic (Fig. 12B). Th. 3 macrosetae a 5e3 , m 4 , m 5p , a 6i , a 7 , and m 7 are absent ( Fig. 12C). If present, m 7 is always a mesoseta. Chaetotaxy of zone Pm of both thoracic segments densely packed with many supplemental macrosetae, forming wing-like patches of posterior setae extending near anterior row, typical for species within the E. bicolor complex (Fig. 3D). Position of pseudopores on Th. 3 atypical for Entomobrya, displaced anteriorly and closer to macrosetae a 1 , a 2 , and a 3 than in other species.
Legs. Trochanteral organ with rectangular setal pattern and up to 37 setae. Unguis with 4 internal teeth; basal teeth enlarged and located approximately middle of inner claw length.
Remarks. Entomobrya bicolor adults can be easily distinguished by the unique color pattern described above combined with the absence of head macroseta Ps 5 (see Table 2 for additional diagnostic characters). Adults have a single, highly stable color form that is easily recognizable and perfectly acceptable for diagnosis (Fig. 11A). Molecular data from Katz et al. (2015) revealed that a small specimen found with E. bicolor, which was completely yellow and lacking all dark pigment, is the juvenile form of E. bicolor (Fig. 11B). The E. bicolor juvenile color form has never been reported in the literature. Unfortunately, it may be difficult to diagnose juvenile members of this species if collected without adults present in the sample. Christiansen (1958b) placed this species in what he termed "the E. bicolor group"; a complex of three closely related species; E. quadrilineata, E. decemfasciata, and E. bicolor. This group is characterized by a highly elongate, cylindrical body, a relatively long furcula, antennae and legs, and by their abundant and highly variable chaetotaxy; many duplicate, supplementary macrosetae, distinctive and augmented setal patterns in thoracic zone Pm, expanded chaetotaxy of Abd. 4, and high levels of asymmetry (Fig. 13A). The extreme setal variation obscures potentially informative characters and results in a lack of discrete, useful diagnostic chaetotaxy between species in this complex. The absence of head macroseta Ps 5 is the only character (other than color pattern) that differentiates E. bicolor from E. decemfasciata and E. quadrilineata. Color pattern is critical for species diagnosis within this complex. See Table 5 for a summary of the important diagnostic characters to separate species in this complex. Etymology. This species is named after the locality it was collected in: Citrus County, Florida. Citrensis is Latin for "from the place of citrus".  Head. Apical bulb of 4 th antennal simple. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈2.5× short setae. Prelabral setae with very fine ciliations that look smooth at low magnification under light microscopy. Ornamentation of the distal margin of the labral papillae with 3-4 spine-like projections on the inner papillae and 2 spine-like projections on the external papillae. Lateral appendage of labial papilla E slightly curved, relatively thin, reaching just below tip of papilla. Dorsal head chaetotaxy slightly reduced (Fig. 15A): macrosetae An' 0 , A 6 , M 3 , M 3i , S' 0 , S 1 , Pi 1 , Pa 2 , Pa 3 , Pm 2 , and Pm 1i always absent; An 3a3 present, asymmetrically absent in one specimen. Eyes G and H small and subequal. Eye patch with 5 setae.
Legs. Trochanteral organ with triangular setal pattern and up to 14 setae. Unguis with 4 internal teeth; basal teeth located approximately middle of inner claw length (Fig. 16A).
Remarks. Entomobrya citrensis sp. n. can be distinguished by its color pattern in combination with the absence of Th. 2 macrosetae m 2 and m 5 , Abd. 2 a 2 and m 3ep , and the presence of Abd. 2 a 3 (see Table 2 for additional diagnostic characters).
This species is closely related to E. assuta; both have highly compressed, or dorsoventrally flattened bodies, reduced chaetotaxy, and similar color patterns. Head macroseta Ps 3 and Abd. 2 macroseta a 3 are both present in E. citrensis sp. n., but are absent in E. assuta. These species can also be separated by color pattern and morphology of the labral papillae (Fig. 2).
See remarks for E. assuta for additional diagnosis information. Only a few specimens of E. citrensis sp. n. were observed from one locality. Additional sampling may reveal more variation in color pattern or chaetotaxy.
Distribution. Endemic to North America. Reported from a single locality: Chassahowitzka National Wildlife Refuge in Citrus County, Florida (Suppl. material 2: D). Guthrie, 1903 Figs 2, 17, 18, 39 Description. Body shape and color pattern. Body dorso-ventrally flattened. Dimorphic color pattern, unrelated to sex (Fig. 17): dark blue, purple, or black pigment covers Th. 3 and Abd. 1-3. Abd. 4 usually white, yellow, or orange with variable levels of pigment ranging from an irregular medial transverse band, randomly distributed irregular patches, or completely pigmented. Th. 2 always white, with a dark band along the anterior margin; medial area of Th. 2 almost transparent in some specimens and internal fat bodies can be observed through the cuticle under a dissecting microscope. Darker specimens with head mostly covered by dark pigment. Antennae usually entirely covered by purple pigment, but some specimens have a mixture of orange, brown, and purple coloration.

Entomobrya clitellaria
Head. Apical bulb of 4 th antennal segment usually simple, sometimes bilobed. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈3× short setae. Prelabral setae ciliate. Ornamentation of the distal margin of the labral papillae with a single seta or spine-like projection (Fig. 2). Lateral appendage of labial papilla E slightly curved, relatively thin, nearly reaching tip of papilla. Labial triangle in one individual with 1 small supplementary ciliate microsetae internal to M1. Dorsal head chaetotaxy ( Fig. 18A) with macrosetae An' 0 , A 3a3 , M 3i , S' 0 , S 6 , and Ps 3 always absent; An 3a2 usually present; Pi 1 , Pm 1 , Pm 1i , and Pp 2 present or absent. Eyes G and H small and subequal. Eye patch with 5 setae.
Legs. Trochanteral organ with triangular setal pattern and up to 26 setae. Unguis with 4 inner teeth; basal teeth located approximately middle of inner claw length.
Remarks. Entomobrya clitellaria can be distinguished by the combination of color pattern, absence of macroseta head S' 0 and Abd. 3 a 1 and presence of head Ps 5 , Th. 2 m 5 , Abd. 2 m 3ep , and Abd. 3 a 2 (see Table 2 for additional diagnostic characters). This species has a relatively conspicuous and diagnostic color pattern, but may be confused with the male E. atrocincta. However, there are obvious differences in their morphology outlined in Table 3. Christiansen and Bellinger (1998) described six different color forms, but after extensive examination of material collected for this study, it was determined that many (but not all) of the color forms they described were most likely variants within a continuous gradient of color pattern variation, without clear genetic isolation (Katz et al. 2015). The two lighter forms (labeled E and F in Christiansen and Bellinger 1998) proved to be elusive and were not collected during this study.
This species is closely related to E. jubata sp. n., but can be easily separated by color pattern, chaetotaxy, and morphology of the labral papillae. The absence of head macrosetae S' 0 and the presence of head macrosetae Ps 5 separate E. clitellaria from E. jubata sp. n. Labral papillae morphology also differs between these species: E. jubata sp. n. has two or three seta or spine-like projection on each papilla, while E. clitellaria only has one seta or spine-like projection per papilla (Fig. 2).
Distribution. Endemic to North America (Suppl. material 2: E).   (Fig. 19). Typical pattern without thoracic bands but with 2-4 irregular, angled bands on lateral margins of abdomen. Color also variable ranging from white, yellow, orange, or sometimes light blue or purple background with black, dark blue, or brown pigment forming bands. Dark bands outlining posterior and lateral margins of Th. 2 and Th. 3 sometimes present. Apex of femora usually with a dark patch. Head either entirely blue or purple, or lacking all pigment except for the eye patches. Dark patches of pigment usually occur on distal end of antennal segments 2-4. Juveniles usually with light blue pigment background and faint brown abdominal banding.
Head. Apical bulb of 4 th antennal segment usually bilobed or simple, but with up to 6 distinct lobes. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈3× short setae. Ornamentation of the distal margin of the labral papillae with single seta or spine-like projection (Fig. 2). Lateral appendage of labial papilla E slightly curved, almost twice as long as papilla. Labial triangle chaetotaxy slightly irregular and atypical for this genus: M1, r, E, L1, L2 ciliate; r significantly smaller than other setae; A1-A5 smooth; sometimes 2 additional ciliate setae inserted internal to M1 and A1, respectively, often relatively difficult to observe. Post labial setae abundant, all type 5. Dorsal head macrosetae (Fig. 20A) An' 0 , An 3a3 , S' 0 , S 6 , Ps 3 , and Pm 1i absent; A 6 sometimes present. Eyes G and H small and subequal. Eye patch with 5 setae. Thorax. Thoracic chaetotaxy developed, highly variable, with many supplemental macrosetae. Th. 2 macrosetae p 6 , p 6e and p 6ep absent. Macrosetae in Zone A so abundant that usually merge with medial macrosetae, forming a single, large patch of setae (Figs 3D; 20B). Th. 3 macrosetae a 5e3 , m 4 , m 5p , a 6i , a 7 , and m 7 absent (Fig. 20C). Macrosetae in Zone Pm also very abundant on both thoracic segments, with many supplemental macrosetae present forming wing-like patches of posterior setae extending near the anterior row (Fig. 3D). Insertion of pseudopore on Th. 3 different from most Entomobrya, displaced anteriorly, very close to macrosetae a 1 , a 2 , and a 3 . Additional duplicate or supplementary setae often form columns internal to a 1 .
Legs. Trochanteral organ with rectangular setal pattern and up to 86 setae. Unguis with 4 internal teeth; basal teeth located approximately middle of inner claw length.
Remarks. Entomobrya decemfasciata can be distinguished by the absence of parallel, longitudinal bands on the thorax, the presence of 2-4 irregular, angled bands on lateral margins of abdomen, and the presence of head macroseta Ps 5 (see Table  2 for additional diagnostic characters). This species belongs to the E. bicolor group (see remarks for E. bicolor), and exhibits high levels of variation in both color pattern and chaetotaxy. E. decemfasciata is perhaps the most setaceous species of Entomobrya reported for North America, clothed in hundreds of macrosetae. This abundant and hyper-variable chaetotaxy provides few characters to differentiate E. decemfasciata from E. bicolor and E. quadrilineata. However, clear differences in color pattern can be observed between these species; E. bicolor lacks band or stripes, E. quadrilineata always has two parallel longitudinal stripes extending from the thorax through Abd. 2 and E. decemfasciata never has bands or stripes on the thorax. Table 5 details important diagnostic characters to separate species within this species complex.
The considerable variation in chaetotaxy and color pattern and relatively high molecular divergences between E. decemfasciata color forms suggest the presence of a cryptic species complex (Katz et al. 2015). However, the lack of diagnostic morphological characters between color forms does not allow the circumscription of new species at this time. More specimens and additional molecular and morphological analyses are needed for further action.
This species has a long history of taxonomic issues (Christiansen 1958b) and its separation from E. quadrilineata only became evident after a thorough molecular analysis (Katz et al. 2015). Christiansen (1958b) separated E. decemfasciata from E. quadrilineata using color pattern, male genital plate, antennal ratios, and later added chaetotaxy in The Collembola of North America (Christiansen and Bellinger 1998). High variability in color pattern and chaetotaxy, deformation of antennae after slide mounting, and the difficulty of discerning the morphology of setae on the genital plate, lessens the utility of these characters for diagnosis. Christiansen and Bellinger (1998) even suggested E. quadrilineata may be a variant form of E. decemfasciata and seemed uncertain regarding differences in chaetotaxy. Furthermore, Christiansen (1958b) reported three distinct color forms for E. decemfasciata (all of which had a V-shape or two angled lateral bands on Abd. 2 and lacking thoracic stripes) and three distinct color forms for E. quadrilineata, one of which also lacks thoracic stripes. During the course of this study, many color forms were collected, including forms both with and without thoracic stripes occurring together in the same sample. These sympatric forms lacked a V-shape or two lateral angled bands on the 2 rd abdominal segment, and, following Christiansen and Bellinger's (1998) concept, were diagnosed as E. quadrilineata. However, large molecular distances made it apparent that forms with and without thoracic stripes were separate species (Katz et al. 2015), warranting further diagnostic inquiry. Type specimens from the Illinois Natural His-tory Survey were attained for both E. quadrilineata and E. decemfasciata. Though both types, preserved in alcohol, were in relatively poor condition, two longitudinal thoracic stripes were clearly observed on the E. quadrilineata specimen (Suppl. material 3: A ,B). Thoracic banding was not observed on the type specimen of E. decemfasciata (Suppl. material 3: C). Based on molecular evidence (Katz et al. 2015) and the observations of types, specimens with the morphology corresponding to both E. quadrilineata and E. decemfasciata can be diagnosed by the presence or absence of parallel thoracic longitudinal stripes. Christiansen's (1958b) report of an E. quadrilineata color form lacking thoracic stripes may have been a case of misidentification of E. decemfasciata. If individuals with and without longitudinal bands were collected together, similar morphology and abdominal pigmentation may have led him to conclude that they were both E. quadrilineata. However, we now know that both species can occur in sympatry.
Distribution. Endemic to North America (Suppl. material 2: F).  Head. Apical bulb of 4 th antennal segment simple. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈5× short setae. Prelabral setae ciliate. Ornamentation of the distal margin of the labral papillae with 2-4 small spine-like projections (Fig. 2). Lateral appendage of labial papilla E slightly curved, thin, nearly reaching tip of papilla. Dorsal head chaetotaxy (Fig. 22A) with macrosetae An' 0 , A 3a2 , A 3a3 , M 3i , S' 0 , S 4i , S 6 , Ps 3 , Pi 1 , and Pm 1i absent; an additional macroseta external to A 3 present in some specimens; S 1 and Pm 1 usually present, but may be asymmetrical. Eyes G and H small and subequal. Eye patch with 5 setae.
Legs. Trochanteral organ with triangular setal pattern and up to 31 setae. Unguis with 4 internal teeth; basal teeth located approximately middle of inner claw length.
Remarks. Entomobrya intermedia can be easily identified by the presence of two longitudinal stripes, a W-shaped mark on Abd. 4 combined with the presence of Th. 2 macrosetae m 5 and Abd. 3 a 1 , and the absence of head macrosetae S 4i , Abd. 2 m 3ep , and Abd. 3 a 2 (see Table 2 for additional diagnostic characters). Historically, this species was considered a synonym of E. nivalis. However, the clear differences in chaetotaxy (see Table 3) and color pattern separate E. intermedia from E. nivalis and other similar forms such as E. multifasciata and female E. atrocincta. Further- more, molecular data supports the separation of this species from E. nivalis (Katz et al. 2015). The combination of chaetotaxy outlined in Table 3 should be used in conjunction with color pattern characters to differentiate this species. Note that observation of chaetotaxy was only examined for specimens collected from Chester County, Pennsylvania. It is likely that specimens from additional localities may re-veal more variation in chaetotaxy than described here, especially since the chaetotaxy reported in Palearctic specimens (Jordana 2012) is different than those observed in North American specimens. Distribution. North America and Europe. The actual distribution of E. intermedia in North America is unclear, as before Christiansen and Bellinger's (1998) monograph the species was included within E. nivalis (Christiansen 1958b Description. Body shape and color pattern. Body cylindrical, slightly dorso-ventrally flattened. Length up to 2 mm. Color pattern monomorphic (Fig. 23): light brown background with black pigment forming dark transverse bands across the posterior margins of Abd. 4, Abd. 5, and Abd. 6; dark pigment present along lateral margins of Th. 2 through Abd. 2, forming two lateral stripes. Two patches of dark pigment usually present medially on Abd. 4 and may appear to form an incomplete irregular transverse band; Th. 2 entirely white except for black pigment lining anterior and lateral margins; legs range in color from white to light brown to purple near the apex.
Head. Apical bulb of 4 th antennal segment usually simple, rarely bilobed. Long differentiated smooth seta on ventral side of 1 st antennal segment 3× as long as short setae. Prelabral setae ciliate. Distal margin of the labral papillae with 2-3 seta or spinelike projections. Labial papilla E with lateral appendage almost straight, reaching tip of papilla. Dorsal head chaetotaxy as in Figure 24A: macrosetae An' 0 , A 3a2 , A 3a3 , M 3i , S 6 , Ps 3 , and Ps 5 always absent; Pi 1 and Pm 1i present or absent; S' 0 always present. Eyes G and H small and subequal. Eye patch with 5 setae. Thorax. Thoracic chaetotaxy well-developed (Fig. 24B). Th. 2 with all described macrosetae present, except m 4i3 . Th. 3 macrosetae a 5e3 , m 4 , m 5p , and a 7 absent (Fig. 24C). Zone Pm with many supplemental macrosetae present in both thoracic segments.
Legs. Trochanteral organ with triangular setal pattern and up to 25 setae. Unguis with 4 inner teeth; basal teeth located approximately middle of inner claw length (Fig. 16B).
Remarks. Entomobrya jubata sp. n. can be easily distinguished by the unique color pattern described above combined with the absence of head macroseta Ps 5 , the presence of head macroseta S' 0 and Th. 2 macrosetae m 2 and m 5 (see Table 2 for additional diagnostic characters). This species is closely related to E. clitellaria and both share similar chaetotaxy. However, these species can be easily separated by color pattern alone; E. jubata sp. n. does not have dark pigment on Th. 3 through Abd.
3. The presence of head macrosetae S' 0 and the absence of head macrosetae Ps 5 also  acterized by the presence of only three microsetae in the eye patch, four macrosetae on Abd. 1, and six macrosetae on Abd. 4. Entomobrya ligata can be separated from E. unifasciata sp. n. and E. neotenica sp. n. by characters outlined in Table 6. E. ligata was described by Folsom (1924) and redescribed by Christiansen (1958b). Both descriptions depict E. ligata with four dorsal transverse bands (Folsom described five bands; he considered the pigment on Abd. 5 and 6 an additional band), two of which occur on the posterior margins of the Th. 2 and Th. 3, respectively. Samples were originally diagnosed as E. ligata based on chaetotaxy described by Christiansen and Bellinger (1998). However, Katz et al. (2015) showed that a population from Chester Co., Pennsylvania was highly divergent and genetically isolated from other populations. The Pennsylvania population differs from all other populations by the presence of a dark transverse band along the posterior margin of Th. 2 . This dark band is present in all individuals collected in Pennsylvania and absent in individuals from all other localities. The original descriptions by Folsom (1924) and Christiansen (1958b) described E. ligata as having this band present and noted a lack of additional variations in color form. Multiple type specimens deposited at the INHS were examined; all collected in the state of New York, and all carry a dark band along the posterior margin of Th. 2 (Suppl. material 3: D-G). Therefore, the combination E. ligata is reserved for populations in which individuals carry the posterior band on Th. 2, whereas populations without this band are referred to E. unifasciata sp. n. (see below). Distribution. Endemic to North America. The species has been reported as having a wide distribution, occurring east of the Mississippi River to the Atlantic coast (Suppl. material 2: I). However, in light of the new circumscription provided above and the possible confusion with E. unifasciata sp. n., most historical reports are questionable, especially those between the western Smokey Mountains and the Mississippi River. The syntypic series of E. ligata was collected at different localities in New York State and all fresh material was collected in Chester Co., Pennsylvania, suggesting the species may be restricted to the northeast section of the country. Description. Body shape and color pattern. Body oval and cylindrical. One primary color form, with slight variations (Fig. 27); yellow background with black, dark brown or purple pigment forming 5 transverse bands along posterior margins of Th. 2 through Abd. 3. Abd. 4 pattern variable, but usually with 2 triangular patches of pigment along posterior margin of segment that point anteriorly toward a W-shaped mark or broken and irregular transverse band. Abd. 5 and Abd. 6 mostly covered with dark pigment. Dark pigment present along lateral margins of all segments, sometimes in broken patches. Antennae light brown or purple pigment increasingly dark towards the apex.
Remarks. Entomobrya multifasciata can be distinguished by the color pattern described above combined with the presence of Abd. 3 macrosetae a 1 and a 2 and the absence of macrosetae head S 4i , Th. 2 m 5 , and Abd. 2 m 3ep (see Table 2 for additional diagnostic characters). Chaetotaxy and color pattern observed in this species is almost indistinguishable from that in female E. atrocincta. Furthermore, variation in chaetotaxy displayed in E. atrocincta obscures most potentially diagnostic characters needed to distinguish E. multifasciata from E. atrocincta. However, E. multifasciata can be recognized by the presence of head macroseta S 4i and the morphology of labral papillae; E. multifasciata has two to three larger seta or spine-like projections per papillae, while E. atrocincta has three to four smaller seta or spine-like projections per papillae (Fig.  2). Table 3 provides additional diagnostic characters separating E. multifasciata from E. nivalis and E. intermedia, which share superficially similar color patterns but can be separated by chaetotaxy. We were unable to obtain North American samples of E. multifasciata and the description and diagnosis provided above are based on specimens from São Miguel Island, Azores, Portugal. The Nearctic distribution of this species remains unclear in light of the sexual dimorphism of E. atrocincta described in this study (see remarks for E. atrocincta). Christiansen and Bellinger (1998) report a widespread distribution, with localities found across the United States, but they also note that their records may be questionable as a result of likely misidentification. Christiansen and Bellinger (1998) describe the labral papillae of E. multifasciata as having three to four small seta or spine-like projections, a condition that resembles those in E. atrocincta, possibly indicating an identification error. In the Collembola of North America, Christiansen and Bellinger (1998) report a male genital plate, presumably from an individual with the E. multifasciata pattern collected in Massachusetts, which eliminates the possibility that the specimen could have been a female E. atrocincta. Since a number of samples were collected in the Northeastern United States (Christiansen and Bellinger 1998), the presence of E. multifasciata in North America cannot be ruled out. However, in the course of the present study, all specimens collected bearing the banded color pattern were female E. atrocincta and were usually accompanied by male E. atrocincta.
Distribution. North America (Christiansen and Bellinger 1998), Hawaii (Christiansen and Bellinger 1992), Europe and Russia (Jordana 2012). Records from North America and Hawaii are questionable due to likely misidentification of E. atrocincta females. See Suppl. material 2: J for a distribution map and below for a list of material examined with collection and locality information.  with black or dark purple pigments forming two lateral triangles on the sides Abd. 3; triangles sometimes reduced to irregular patches. Additional irregular patches of pigment usually on lateral margins of all segments. A band sometimes present along posterior margin of Th. 2. Abd. 5 with 2 dark spots, sometimes forming irregular triangular shapes. Antennae usually light purple near apex and relatively long. Legs usually white, with small purple patches on apical end of femora.
Head. Apical bulb of 4 th antennal segment simple or bilobed. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈2-2.5× short setae. Four prelabral setae finely ciliate, appearing smooth under light microscopy. Ornamentation of the distal margin of the labral papillae with single seta or spine-like projections (Fig.  2). Labial lateral appendage slightly curved, relatively thick, not reaching tip of papilla. Dorsal head chaetotaxy reduced (Fig. 30A), macrosetae An' 0 , An 3a2 , An 3a3 , A 6 , M 3i , S' 0 , Ps 3 , Pi 1 , Pi 2 , Pm 1 , Pm 1i , and Pp 2 absent; S 5i usually present; S 4i and S 6 usually absent. Eyes G and H smaller than A-F but enlarged; G slightly larger than H. Eye patch with 3 setae.
Abdomen. Abdominal chaetotaxy reduced; no macrosetae variation observed. Abd. 1 with 4 macrosetae; a 5 , m 2 , m 3 , and m 4 (Fig. 30D). Abd. 2 with 4 macrosetae: a 2 , m 3 , m 3e , and m 5 (Fig. 30E). Abd. 3 with 3 macrosetae: m 3 , pm 6 , and p 6 (Fig. 30F). Abd. 4 with 6 inner macrosetae (Fig. 30G). Mucronal teeth subequal; mucronal spine smooth. Remarks. Entomobrya neotenica sp. n. can be diagnosed by the presence of two lateral dark triangular shaped or irregular spots on Abd. 3, only 3 setae in eye patch, and the absence of head mesoseta An' 0 and Abd. 3 macroseta m 3ep (see Table 2 for ad-ditional diagnostic characters). This species is included in the E. ligata complex (see remarks for E. ligata) and has a unique color pattern and chaetotaxy never before reported in the literature. E. neotenica sp. n. is exceptionally small compared to most Nearctic Entomobrya. In fact, most individuals were thought to be juvenile forms of E. ligata prior to the observation of the male genital plate. Entomobrya neotenica sp. n. is very similar to E. ligata and E. unifasciata sp. n., but can be separated by characters outlined in Table 6.

Entomobrya nivalis (Linnaeus), 1758
Figs 2, 31, 32, 39 Description. Body shape and color pattern. Body cylindrical. One primary, but variable, color form ( Fig. 31): yellow or white background with black, dark brown or purple pigment always forming thin transverse bands along the posterior margin of Th. 3, and Abd. 2 through Abd. 6. Additional transverse bands present or absent on Th. 2 and Abd. 1. Abd. 4 usually with U-shaped or "11"-shaped pattern connecting basally with band along posterior margin. Antennae usually lack dark pigmentation, sometimes with light brown or purple pigment, darkening near the apex.
Legs. Trochanteral organ with triangular setal pattern and up to 34 setae. Unguis with 4 internal teeth; basal teeth located approximately 60% of inner claw length. Unguiculus acuminate with small serrations on internal edge.
Abdomen. Abd. 1 with 7-10 macrosetae (Fig. 32D). Abd. 2 macroseta a 2 , a 3 , m 3 , m 3e , m 3ep , and m 5 present; m 3ei and m 3ea usually absent (Fig. 32E). Abd. 3 macroseta a 1 , m 3 , am 6 , pm 6 , and p 6 present (Fig. 32F); a 3 usually absent. Abd. 4 with 7-8 inner macrosetae (Fig. 32G). Mucronal teeth subequal. Remarks. Entomobrya nivalis can be diagnosed by the presence of a U-shaped or "11" shaped pattern on Abd. 4 combined with the presence of macrosetae head S 4i , Th. 2 m 5 , Abd. 2 m 3ep , and Abd. 3 a 1 , and the absence of Abd. 3 a 2 (see Table  2 for additional diagnostic characters). This species has a highly variable color pattern with many intermediate forms that intergrade with E. atrocincta females, E. intermedia, and E. multifasciata. However, E. nivalis can be separated from these species by chaetotaxy (Table 3) and, with careful consideration, color pattern; the presence of a U-shaped or "11" shaped pattern on Abd. 4 is unique to this species. Therefore, it is critical to evaluate chaetotaxy in addition to color pattern when making a species diagnosis. It is important to note that the large genetic distances between presumably conspecific individuals (Katz et al. 2015;Feng Zhang, personal communication), differences in chaetotaxy between populations in North America and Europe (Jordana 2012), and variable color pattern among populations across its world-wide distribution, suggests that E. nivalis likely represents a cryptic species complex.
Distribution. North America and Europe. Records of E. nivalis in North America are suspect if diagnosed without considering chaetotaxy given the superficial similarities in color form expressed by E. atrocincta females (See Fig. 9G,H). See Suppl. material 2: L for a distribution map and below for a list of material examined with collection and locality information.

Entomobrya quadrilineata
Head. Apical bulb of 4 th antennal segment usually bilobed. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈4× short setae. Four prelabral setae ciliate. Ornamentation of the distal margin of the labral papillae with single seta or spine-like projection (Fig. 2). Lateral appendage of labial papilla E almost twice as long as papilla. Labial triangle chaetotaxy slightly irregular and atypical: M1, r, E, L1, L2, all ciliate; r significantly smaller than other setae; a supplementary ciliate seta sometimes present internal to M1, and relatively difficult to observe; A1-A5 smooth. Dorsal head chaetotaxy (Fig. 34A) with macrosetae An' 0 , An 3a3 , A 6 , S' 0 , S 6 , Ps 3 , and Pm 1i absent; Pi 1 sometimes present. Eyes G and H small and subequal. Eye patch with 5 or 6 setae.
Thorax. Thoracic chaetotaxy greatly developed, with high levels of variation and many supplemental macrosetae. Th. 2 zone A enlarged and sometimes merging with medial macrosetae forming a single, large patch of setae (Fig. 34B). Th. 3 macrosetae m 4 , m 5p , a 6i , and a 7 absent (Fig. 34C). Both thoracic segments with zone Pm enlarged, with many supplemental macrosetae present forming wing-like patches and extending near anterior row (Fig. 34B,C). Th. 3 pseudopore displaced anteriorly, very close to macrosetae a 1 , a 2 , and a 3 . Legs. Trochanteral organ with rectangular setal pattern and up to 41 setae.
Type  (35.57030,-83.16917), under bark, 29.v.2011, AK11-39;11 in vial, North Carolina, Swain Co., Great Smoky Mountains National Park, Balsom Mountain, Heintooga Ridge Rd. (35.57030,-83.16917 Description. Body shape and color pattern. Body oval and cylindrical. Length up to 1.85 mm. Color pattern stable (Fig. 35), always with 3 transverse bands, 1 thin regular band along posterior margin of Th. 3, an irregular, patchy, thick band covering most of Abd. 3, and a highly irregular and sometimes broken band across medial section of Abd. 4. A small patch of pigment covers Abd. 5 and 6. Patterns usually consisting of black or dark blue pigment on a yellow background. Dark pigment usually occurring in patches along lateral margins of Th. 2 through Abd. 4. Small, rectangular black patches may occur in pairs on posterior margin of Th. 2, Abd. 1, and Abd. 2. A faint transverse band sometimes on posterior margin of Th. 2, but if present, always much lower in opacity compared to transverse band along posterior margin of Th. 3. Antennae with purple pigment, darker near apex. Legs usually white, with small purple patches on apical end of femora. A medial ring of purple pigment also occurs on tibiotarsus of hind legs.
Head. Apical bulb of 4 th antennal segment usually bilobed, sometimes simple. Long differentiated smooth setae on ventral side of 1 st antennal segment ≈3× short setae. Four prelabral setae finely ciliate, seemingly smooth at low magnification under light micros- copy. Ornamentation of the distal margin of the labral papillae with single seta or spine-like projection (Fig. 2). Lateral appendage of labial papilla E short, extending only ¾ papilla length. Dorsal head chaetotaxy ( Fig. 36A) with macrosetae A 6 , M 3i , S 6 , Ps 3 , Pi 1 , Pm 1i , and Pp 2 , absent; S' 0 usually absent, but observed in 2 individuals; An' 0 , a short mesoseta present medially between both An 0 . Eyes G and H small and subequal. Eye patch with 3 setae.
Legs. Trochanteral organ with triangular setal pattern and up to 22 setae. Unguis with 4 internal teeth; basal teeth located approximately middle of inner claw length (Fig. 16D). Unguiculus acuminate with small serrations on internal edge.
Remarks. Entomobrya unifasciata sp. n. can be diagnosed by the presence of only three dark transverse bands (no band across the posterior margin of Th. 2), presence of head mesoseta An' 0 , 4 macrosetae on Ab. 1, and 3 eye patch setae (see Table 2 for additional diagnostic characters). This species is part of the E. ligata complex (see remarks for E. ligata) and is very similar to E. ligata and E. neotenica sp. n., but can be separated by characters outlined in Table 6. Though identical in chaetotaxy, molecular evidence (Katz et al. 2015) and the absence of a dark transverse band on the posterior margin of Th. 2 separate this species from E. ligata.
Distribution. Endemic to North America (Suppl. material 2: N). Many records of E. ligata, especially those collected from the Smokey Mountain region west to the Mississippi River, are likely to be E. unifasciata sp. n.  Stach, 1930 Figs 2, 3C, 37, 38, 39 Description. Body shape and color pattern. Body relatively robust and cylindrical. Color form largely monomorphic (Fig. 37)    (COI and morphology). Entomobrya species groups are highlighted with colored boxes. Branches of representing taxa from the same species were collapsed into triangles (triangle length represents branch lengths between collapsed branches) for simplicity. Branch labels include posterior probabilities (above) and maximum likelihood bootstrap support (below). Scale bars represent 0.2 base substitutions. also with members in the genus Homidia, but not with other Entomobrya treated here. Entomobrya unostrigata is a recently introduced species, now with a widespread Nearctic distribution (Christiansen and Bellinger 1998), which may explain its distinction from other North American Entomobrya species included in the present study. The E. unostrigata specimens observed for this study have only two large seta or spine-like projections on each labral papillae (Fig. 2). However, multiple variations of the labral papillae have been reported (Christiansen 1958b;Christiansen and Bellinger 1992;Christiansen and Bellinger 1998;Jordana 2012

Morphological phylogeny
Bayesian analysis of 179 morphological characters from 23 taxa produced a single consensus tree with high support (Fig. 39B). The most likely tree inferred by the maximum likelihood analysis is congruent with the Bayesian tree and bootstrap values were added to the Bayesian consensus tree (Fig. 39B). When Pseudosinella violenta is designated as the outgroup, the topology of the tree assumes a ladder-like evolutionary progression that is associated with an increase in the number of dorsal macrosetae. Entomobrya assuta and E. citrensis sp. n., the two species with the smallest number of macrosetae, form a monophyletic group at the base of the tree, whereas Entomobrya bicolor, E. decemfasciata, and E. quadrilineata, which are characterized by an abundance of dorsal macrosetae, form the most derived monophyletic group. This tree indicates that Entomobrya is paraphyletic, as the genera Seira, Homidia, Entomobryoides, and most representatives of Willowsia are interspersed among clades of Entomobrya species. The two species in the genus Homidia form a monophyletic group, but the genus Willowsia is not monophyletic. Other clades identified by the molecular COI analysis in Katz et al. (2015) (Fig. 39A) were not resolved as monophyletic groups based on morphological characters.

Combined phylogeny (COI and morphology)
Bayesian analysis based on combined morphology and COI (Katz et al. 2015) datasets yielded a highly supported tree (Fig. 39C). The most likely tree inferred by the maximum likelihood analysis is congruent with the Bayesian tree and bootstrap values were added to the Bayesian consensus tree (Fig. 39C). The addition of molecular characters resolves all monophyletic species groups/clades of Entomobrya observed in the COI only tree (Fig. 39A). However, the deeper relationships among clades follow the progressive evolution towards an increased number of macrosetae supported by the morphological analysis. Both analyses (morphology only and combined) indicate that Entomobrya is paraphyletic.

Diagnostic and phylogenetic utility of chaetotaxy
Detailed examination of the adult chaetotaxy of 15 species of North American Entomobrya suggests that the exclusive use of chaetotaxy for species diagnosis and as phylogenetic characters can potentially cause serious confusion. This study uncovered high levels of previously undocumented intraspecific variation and asymmetry of dorsal macrosetae (Fig. 13), and has made it clear that examination of many individuals is critical to properly identify variable chaetotaxy in order to choose appropriate characters for species delimitation. For example, Christiansen and Bellinger (1998) separate many species based on the chaetotaxy of Abd. 2 and Abd. 3, but in some species groups both regions contain significant intraspecific variation. In the Collembola of North America (Christiansen and Bellinger 1998), species within the nivalis group (E. nivalis, E. atrocincta, E. multifasciata, and E. intermedia) are separated by a combination of color pattern and by the presence or absence of four macrosetae: m 3ep on Abd. 2 and a 1 , a 2 , and a 3 on Abd. 3. This study shows that these characters must be used in combination to provide sufficient separation of these species due to overlapping intergrades of color forms and the presence macrosetae polymorphisms. Species in the bicolor group (E. bicolor, E. decemfasciata, and E. quadrilineata) present the most obvious example of the failure of chaetotaxy to provide an adequate means for species separation. Large numbers of macrosetae, extreme levels of inter-individual variation, and the common occurrence of asymmetries, cause significant overlap and obscure the homology of macrosetae otherwise considered important in species separation. The present study shows that color pattern is absolutely critical for the identification of species within this group.
These problems are cause for concern considering the important role of dorsal chaetotaxy in diagnosis and delimitation of species in the family Entomobryidae. Many studies are based on chaetotaxy as primary (or sole) evidence for species separation, following Szeptycki's (1979) nomenclatural system (Chen and Christiansen 1993;Jordana 2012). The homology of macrosetae as defined by Szeptycki (1979) assumes that a fixed number of macrosetae occur in strict, predefined positions rather than randomly distributed within a given area. Homologies are easier to determine between species in genera characterized by small numbers of macrosetae, and chaetotaxy likely provides accurate phylogenetic estimation in these groups. However, in groups such as Entomobrya, intraspecific variation, differences in setae arrangements, asymmetries, and large numbers of supplemental setae, render homology assessment a subjective and arbitrary process (Potapov and Kremenista 2008). Incorrect homology assignments obscure any useful phylogenetic information provided by chaetotaxy.
Post-embryonic studies that test Szeptycki's (1979) hypotheses have been successful in identifying and refining setae homologies in some pluri-and polychaetotic species and groups (Soto-Adames 2008; Zhang et al. 2011), but these studies are laborious and are only conclusive for species and/or groups examined. Information regarding the post-embryonic development of Entomobrya chaetotaxy is very limited and mainly concerned with Palearctic species (Szeptycki 1979). Explicit hypotheses concerning the underlying molecular mechanisms governing macrosetae development and position, to our knowledge, have only been tested in Diptera. These mechanisms are generally very complex and highly regulated processes, controlled by multiple genes that ensure macrosetae do occur in strictly defined locations (Simpson 1990;Heitzler et al. 1996;Leyns et al. 1996;Simpson et al. 1999;Furman and Bukharina 2008). However, it is uncertain (and unlikely) that Collembola macrosetae, and/or the genes that regulate them, are homologous to those of Diptera.
The phylogenies based on morphological and molecular data illustrate the effects that chaetotaxy has on phylogeny estimation. The trend of progressive evolution towards an increased number of macrosetae presented in the morphology and combined phylogenies is suspicious and may be driven by outgroup choice and character coding strategy. Entomobrya assuta and Entomobrya citrensis sp. n., sister species characterized by reduced chaetotaxy, are basal to all other Entomobrya, together with all scaled species included in the analysis (Pseudosinella, Willowsia, and Seira), which also have few macrosetae. This association is not reflected in the analysis of COI alone (Katz et al. 2015;Fig. 39A) and is likely due to the accumulation of characters (each seta in a multiplet was defined and scored as a separate character, see Suppl. material 1) present in polychaetotic species but absent in the E. assuta group and the outgroup. Evaluation of coding strategies for chaetotaxy, and the effect of outgroup choice is clearly needed to exact the maximum amount of phylogenetic information while minimizing homoplasy. Despite the conflict in branching pattern for deep nodes estimated by morphology and COI, the combined analysis retains the species composition and relationship of the five species groups identified by the COI analysis. These relationships are quite obvious and were proposed by Christiansen (1958b) before modern sequencing and dorsal chaetotaxy systems were available.

Phylogenetic relationships within Entomobryini and Willowsia
The result of the combined phylogenetic analysis (COI and morphology) of 15 species of North American Entomobrya in addition to 6 species representing 3 closely related genera (Entomobryoides, Homidia and Willowsia) and 2 outgroup species (P. violenta and S. dowlingi), generated a highly supported phylogeny of the North American Entomobryini and Willowsia. Several interesting results were observed concerning relationships among some currently recognized Entomobrya species. Entomobrya nivalis, E. intermedia, E. multifasciata, and E. atrocincta were resolved as a monophyletic clade; all are closely related but distinct species diagnosable using morphology and COI sequences. Overlapping intraspecific variation in color pattern and chaetotaxy has caused many to consider these species synonymous (See Christiansen 1958b and Jordana 2012 for revision history). This study has, for the first time, combined substantial molecular (Katz et al. 2015) and morphological evidence in support of their separation. It is also worth noting that these were the only Entomobrya species included in the analysis with Holarctic distribution, all commonly reported throughout North America (Christiansen and Bellinger 1998) and Europe (Jordana 2012). The monophyly of the group and separation from species endemic to North America suggest a common origin in Europe or Palearctic region. It is possible that the group is either part of a shared, relictual Laurasian fauna or that the species were introduced from Europe in historical times. Evaluation of these two hypotheses will require molecular analysis of extensive samples of North American and European populations.
The resulting phylogeny also raises questions regarding the generic relationships within the tribe Entomobryini (here represented by the genera Entomobrya, Entomobryoides and Homidia) and Willowsia, a closely related genus. Research concerning the systematics of these groups is limited, and most phylogenetic studies have focused on suprageneric relationships by utilizing morphology, allozymes, and/or ribosomal markers that provided limited resolution and support of generic relationships (Lee and Park 1991;Lee et al. 1995;D'Haese 2002;Xiong et al. 2008). However, more recent work by Zhang et al. (2014b) indicated the non-monophyly of Entomobryini and Willowsiini based on molecular phylogeny. Zhang et al. (2014a) also questioned the monophyly of Willowsiini. Further analyses of S-chaetae (dorsal sensilla) dismissed Willowsiini as monophyletic (Zhang and Deharveng 2015).
This study supports Zhang et al.'s (2014aZhang et al.'s ( , 2014b and Zhang and Deharveng's (2015) findings regarding the non-monophyly of Entomobryini and Willowsiini. Our phylogenetic analysis places both Willowsia nigromaculata (Lubbock) and W. buski (Lubbock) within the Entomobrya clade, further supporting the paraphyly of Entomobrya. This is not entirely unexpected considering the means by which these genera are differentiated. The genus Entomobrya is considered the most morphologically generalized group of Entomobryinae without distinct apomorphies while morphologically similar species with autapomorphic characters are separated into different genera (e.g., Willowsia with scales, Entomobryoides with smooth tibiotarsal setae and Homidia with dental spines). In light of the relationships inferred from the phylogeny, the classification of Entomobrya, along with some other genera within Entomobryidae, may need to be reevaluated in the future.
Results also indicate that the genus Willowsia may be polyphyletic. A new Willowsia species (Willowsia n. sp. 1) collected in Citrus Co., Florida, is resolved as a sister species to the Entomobrya clade, while W. buski and W. nigromaculata seem to have evolved from lineages within the Entomobrya clade. Considering their world-wide distribution, presumably spread by humans, and their similarity to Asian species (Zhang et al. 2011), W. buski and W. nigromaculata most likely were introduced into North America from Asia. However, Willowsia n. sp. 1 shares characteristics (i.e., scale type; see Zhang et al. 2011) with W. mexicana Zhang, Palacios-Vargas & Chen, the only other Willowsia species known to be endemic to North America (Zhang et al. 2007). Willowsia n. sp. 1's morphological similarity to W. mexicana may support an inde-pendent origin of New World Willowsia. Further exploration by utilizing additional markers and more complete taxon-sampling among closely related genera is needed in order to establish appropriate generic relationships and classifications.