Molecular Evaluation of the Fairy Shrimp Family Branchinectidae (Crustacea: Anostraca) Supports Peripatric Speciation and Complex Divergence Patterns

© 2020 Academia Sinica, Taiwan Open Access Molecular Evaluation of the Fairy Shrimp Family Branchinectidae (Crustacea: Anostraca) Supports Peripatric Speciation and Complex Divergence Patterns D. Christopher Rogers1,* and Andres Aguilar2 1Kansas Biological Survey, Kansas University, Higuchi Hall, 2101 Constant Avenue, Lawrence, KS 66047-3759 USA. *Correspondence: E-mail: [email protected] (Rogers) 2Department of Biological Sciences, California State University, 5151 State University Drive Los Angeles, CA 90032 USA. E-mail: [email protected] (Aguilar) Received 13 August 2019 / Accepted 21 February 2020 / Published 28 April 2020 Communicated by Benny K.K. Chan The Branchinectidae is a diverse and widely distributed group of anostracans. The majority of work on the group has focused on the morphological delineation of taxa and biogeography. Here we present a molecular phylogeny for select members of the family to better understand the distribution of morphological variation among species, and test biogeographic models of speciation for the group. Although we conducted both molecular and morphological phylogenies for the Branchinectidae, the morphological analysis did not support our molecular phylogeny and it did not support previous species group concepts based on geography. Our molecular phylogenetic analysis suggests that the family may have originated in Eurasia and found support for numerous species groups. These phylogenetic groups assisted in delineating species groups that are all definable morphologically and/or ecologically. The peripatric speciation model was supported from our analysis, offering credence to previously published speciation models in anostracans. This suggests that these processes may be important in other Branchiopoda and should be rigorously evaluated when delineating species. Key words: Branchinecta, Archaebranchinecta, Species groups, Phylogeny, Branchiopoda. Citation: Rogers DC, Aguilar A. 2020. Molecular evaluation of the fairy shrimp family Branchinectidae (Crustacea: Anostraca) supports peripatric speciation and complex divergence patterns. Zool Stud 59:14. doi:10.6620/ZS.2020.59-14. BACKGROUND Anostracan crustaceans are among the least known of all faunal elements in seasonally astatic aquatic habitats, yet they are used as indicators of ephemeral wetland habitat health and functionality in the United States (Rogers 1998; Lang and Rogers 2002), Brazil (Rogers and Ferreira 2007) and Australia (M. Coleman & B. Datsun personal communication; B. Timms personal communication). For example, of the 26 anostracan species reported from California, only two were known prior to 1980, and eight were not described until after 1990. Large branchiopods have become flagship animals for ephemeral wetland habitat imperilment and are the subject of increasing conservation attention (Federal Register 1994; Eriksen and Belk 1999; Brendonck et al. 2008). Currently, five large branchiopod crustacean species are afforded protection under the federal Endangered Species Act, and other species are proposed for protective status. Documentation of the diversity of anostracan crustaceans and their associated habitats can identify trends in habitat and species loss relative to land use practices. Effective conservation management of anostracan crustaceans requires knowledge of their phylogeny, taxonomy, distribution and habitat requirements. Zoological Studies 59:14 (2020) doi:10.6620/ZS.2020.59-14 1 © 2020 Academia Sinica, Taiwan The Branchinectidae is comprised of two extant genera: Archaebranchinecta, with three species, and Branchinecta, with 51 species (Rogers 2013; Cohen et al 2019). The species of Archaebranchinecta are limited to the Americas (one fossil in North America, two extant in South America) whereas Branchinecta is distributed through the Holarctic and the Neotropic regions, with one species occurring in the northernmost Antarctic (Rogers 2013; Rogers and Coronel 2011). Branchinecta has two primary centres of diversity; one in western North America (29 species) and one in southern South America (16 species) (Rogers 2006), with the remaining six species in Eurasia. Of these 51 species, 31 species are narrow range endemics, 12 species are known from two or less localities each, and four species are protected under the USA Endangered Species Act (Eng et al. 1990; Fugate 1993; Belk and Fugate 2000; Rogers and Fugate 2001; Belk and Rogers 2002; Rogers 2006; Rogers et al. 2006 2011; Rogers and Lorenz 2015). The reason for the high western North American endemism is the complex geochemical and recent geological history of this region (Rogers 2014a 2015). Colonization of newly available habitats (e.g., exposure of ‘new’ terrestrial environments via uplift or erosion) and/or novel environments (e.g., saline versus alkaline pools) may be important for driving speciation in the Branchinectidae and allied anostracans (Rogers 2015). Additionally, Branchinecta is the most diverse anostracan genus in South America, further suggesting that this group is the prime system to study not only in situ continental divergence, but South American adaptive radiations. The monophyly of Branchinectidae has been strongly supported by recent phylogenetic analyses (e.g., Remigio and Hebert 2000; Weekers et al. 2002), but relationships within the family have not been clearly elucidated. Morphological diagnosis of anostracan species is generally straightforward as there is strong sexual selection by the females for large and showy male second antennae and associated structures. The most reliable characters used to delineate anostracan species are the form of the male second antennae (Lynch 1972). Other characters, such as the form and/ or ornamentation of the female head, dorsum, and brood pouch, the structure and shape of the male gonopods, and the external morphology of the eggs are of limited use at the species level (Lynch 1972; Rogers and Fugate 2001; Belk and Rogers 2002; Rogers 2002 2006; Rogers et al. 2006). These features may display a large amount of homoplasy and be inappropriate for determining phylogenetic relationships among anostracan groups. In recent molecular phylogenetic studies of the Australian Branchinella (Remigio et al. 2003; Pinceel et al. 2013), it was found that only one of the morphologically defined groups described by Geddes (1981) was monophyletic, and all others were polyphyletic. Similarly, the morphological features described by Hamer et al. (1994a b) and MaedaMartinez et al. (1995) proved to be homoplastic based on the molecular phylogeny of Streptocephalus (Daniels et al. 2004). A detailed analysis of a single species complex (Branchinella longirostris Wolf, 1911) found no relationship between frontal appendage morphology and molecular relatedness (Zofkova and Timms 2009). These studies indicate that the morphological features commonly used to differentiate the various anostracan species may not be that reliable for determining evolutionary relationships, although other characters may exist that will provide more information than previously thought. Therefore, independent molecular phylogenetic analysis is warranted to establish closely related subgeneric groups and serve as a scaffold by which to better understand anostracan species level morphological differentiation. We initiated this study to gain a better understanding of the evolutionary relationships among members of the Branchinectidae. Our initial goal was to evaluate if morphologically similar species within the family are in fact closely related from a molecular phylogenetic perspective. We simultaneously investigated the phylogenetic utility of a suite of morphological features, alone and in combination with molecular data. The phylogenetic hypotheses generated from molecular and morphological data were then used to assess biogeographic hypotheses for the group and assess the systematics of the family. MATERIALS AND METHODS Specimen Collection We examined the morphology of all 54 branchinectid species. Character states of seven species were only available via the literature; however, the remainder were examined directly. We examined 1,727 male and 1,801 female branchinectid fairy shrimp from both described genera (3,528 individuals total). The specific collecting data of the material used in this study are presented in appendix 1. Thirty specimens from 29 species were sequenced for the molecular study. Specimens were either adults preserved in 95% ethyl alcohol or reared from eggs in the laboratory. For each population that was reared from eggs, we collected soil from natural, dried field sites. We made soil collections by sampling at many locations across each dried habitat and then homogenizing the soil in plastic bags. Approximately 500 mL (volume) of page 2 of 17Zoological Studies 59:14 (2020) © 2020 Academia Sinica, Taiwan this field-collected soil was placed in the bottom of a 37 L aquarium and hydrated with deionized water. The aquarium was maintained at 25–28°C, with gentle aeration, 12 hours light/dark cycle, and fed a mixture of brewer’s yeast and ground vegetable flake fish food. Laboratory Analysis Preserved specimens were examined using a Wild M8 dissection stereomicroscope. Some preserved specimens have been in storage over 10 years, making amplification of large fragments via the polymerase chain reaction (PCR) difficult. Members from the Chirocephalidae (Eubranchipus oregonus and Linderiella occidentalis – Table 1) were used as outgroup taxa, following previous phylogenetic analyses (Remigio and Hebert 2000; Weekers et al. 2002). DNA was extracted from individual samples with a modified CTAB extraction protocol for anostracans (Aguilar 2011). Samples were macerated in 2x CTAB buffer (Teknova) and 10 μL of Proteinase K (10 mg/mL) and allowed to incubate overnight at 60°C. Following incubation samples were extracted once with PhenolChloroform-Isoamyl alcohol (25:24:1) and once with Chloroform-Isoamyl Alcohol (24:1). DNA was then precipitated overnight in ethanol (with 3 μL of 3 M NaOH) at -20°C. DNA was pelleted via centrifugation, washed once with 70% ethanol, and allowed to air dry. The DNA pellet was resuspended in 100 μL on sterile ddH2O. All DNA extractions were quantified on a Nanodrop 1000 spectrophotometer. We targeted three genes for phylogenetic evaluation: two mitochondrial (16S and 12S) and a single nuclear locus (28S). Primer sequences and sources are listed in table 2. All reactions were conducted in 30 μL volumes with 1x PCR Buffer II Table 1. Specimens used in phylogenetic analysis and corresponding GenBank Accession Numbers Species Sample location 16S 12S 28S Ingroup taxa B. campestris Carrizo Plains, CA, USA MT010634 MT010665 MT010695 B. coloradensis Ada, Idaho, USA MT010635 MT010666 MT010696 B. conservatio Solano, CA, USA MT010635 MT010667 MT010697 B. constricta Fremont, WY, USA MT010637 MT010668 MT010698 B. cornigera Grant, WA, USA MT010638 MT010669 MT010699 B. dissimilis Modoc, CA, USA MT010638 MT010670 MT010700 B. ferox Jordan MT010640 MT010671 MT010701 B. gaini Antarctic Peninsula MT010641 MT010672 MT010702 B. gigas Washoe, NV, USA MT010642 MT010673 MT010703 B. granulosa Chile MT010643 MT010674 MT010704 B. hiberna Humboldt, NV, USA MT010644 MT010675 MT010705 B. lateralis Wyoming, USA MT010647 MT010678 MT010708 B. lindahli-1 Mineral, NV, USA MT010648 MT010679 MT010709 B. lindahli-2 Lancaster, CA, USA MT010649 MT010680 MT010710 B. longiantenna Carrizo Plains, CA, USA MT010650 MT010681 MT010711 B. lutulenta Grant, WA, USA MT010646 MT010677 MT010707 B. lynchi Merced, CA, USA MT010651 MT010682 MT010712 B. mackini Washoe, NV, CA MT010652 MT010683 MT010713 B. mediospinosa Kansas, USA MT010645 MT010676 MT010706 B. mesovallensis San Joaquin, CA, USA MT010653 MT010684 MT010714 B. oriena Douglas, NV, USA MT010654 MT010685 MT010715 B. orientalis Turkey MT010655 MT010686 MT010716 B. oterosanvicentei Baja California, MX MT010656 MT010687 MT010717 B. packardi Cibola, NM, USA MT010657 MT010688 MT010718 B. paludosa Manitoba, CAN MT010658 MT010689 MT010719 B. potassa Nebraska, USA MT010659 MT010690 MT010720 B. raptor Elmore, ID, USA MT010660 MT010691 MT010721 B. sandiegonensis San Diego, CA, USA MT010661 MT010692 MT010722 B. serrata Fremont, WY, USA MT010662 MT010693 MT010723 Outgroup taxa Eubranchipus oregonus Siskiyou Co., CA, USA MT010664 - MT010725 Linderiella occidentalis Merced, CA, USA MT010663 MT010694 MT010724 page 3 of 17Zoological Studies 59:14 (2020) © 2020 Academia Sinica, Taiwan (ABI), 2.0 mM MgCl2, 0.08 mM of each dNTP, 0.4 μM of each primer and 1 unit of Amplitaq (ABI). Thermal cycling was performed on an ABI3700 thermocycler with the following temperature profile: initial denaturation for 5 min at 94°C followed by 30–35 cycles at 94°C for 30 s, primer specific annealing temperature (Table 2) for 30 s and an extension at 72°C for 45 s, followed by a 5 min extension at 72°C. PCR products were run out on a 2% agarose gel to check for amplification prior to sequencing. All PCR products were cleaned with the EXO-SAPIT kit (USB) and sequenced in both directions on an Applied Biosystems 3730 automated sequencer. We were only able to acquire genetic samples from just over half (55%) of the extant species, as sampling in South America was limited. Phylogenetic analysis Sequence trace files were imported into SEQUENCHER (Genecodes Inc.), and inspected by eye for read quality. Consensus reads were constructed for each individual/locus combination and exported to MUSCLE (Edgar 2004) for alignment using default alignment parameters. Sequence evolution models were evaluated for each gene fragment and for the concatenated dataset with the Bayesian Information Criterion (Schwartz 1978) in jMODELTEST v1.10 (Posada 2008). A total of 51 morphological characters (section 4.1.) were coded for all species that we were able to obtain molecular data for, including outgroup taxa (matrix in Table S1). MrBayes v3 (Ronquist and Huelsenbeck 2003) was used to reconstruct phylogenies in a Bayesian framework, and the dataset was partitioned based on the three gene fragments (12S, 16S, 28S) or the three gene fragments and the morphological data. We used the standard discrete model (Lewis 2001) with gamma rate variation for the morphological partition in the Bayesian analysis. Four simultaneous runs of six MCMC chains were run for a total of 5 × 106 generations, sampling trees every 100 generations for a total of 50,000 trees. The first 25% of trees (12,500) were discarded as burn in. A 50% consensus tree was estimated from the remaining sampled trees to better show the conflicts. Lastly, we performed a phylogenetic analysis of just the 51 coded morphological features. This analysis was done in PAUP4 (Swofford 1997) using a heuristic search and no character weighting. We ran 1,000 bootstrap replicates to assess node confidence. List of morphological characters 1. Female second antenna (AII) medial surface [0] smooth; [1] medial spine; [2] lateral bulge; [3] medially chitinized. 2. Female AII length: [0] normal; [1] elongate. 3. Male AII extending to: [0] thoracic segment (TH) IV; [1] Th V; [2] Th VI; [3] Th VIII; [4] Th XII. 4. AII proximal vs distal antennomere length: [0] prox < dist; [1] prox = dist; [2] prox > dist. 5. Antennal appendages: [0] none; [1] anteriobasal. 6. Pulvilli: [0] absent; [1] present. 7. Apophyses: [0] flat; [1] cylindrical; [2] transverse; [3] conical. 8. Medial AII spines: [0] microdenticles only; [1] small scattered; [2] small, in rows; [3] generally distributed; [4] large, in discrete row(s); [5] on medial projection. 9. Anterior AII spines: [0] absent; [1] present. 10. Medial AII surface: [0] smooth; [1] convex; [2] ridged; [3] hooked. 11. Medial AII denticles: [0] microdenticles only; [1] generally distributed; [2] systematic field. 12. AII distal antennomere: [0] subcylindrical; [1] anteriorly flattened; [2] narrowly laterally flattened; [3] broadly laterally flattened; [4] triangular in cross section, smooth. 13. AII distal antennomere torsion: [0] none; [1] anteriorly. 14. AII Distal antennomere arc: [0] none; [1] present. 15. AII Distal antennomere arc: [0] proximal; [1] medial; [2] even; [3] distal. 16. AII apex: [0] subacute; [1] rounded; [2] anteriorly produced; [3] truncated. 17. AII subapical constriction: [0] absent; [1] present. Table 2. PCR primers used in this study Primer Sequence (5' to 3') Annealing temperature Reference 16Sbr-3' CCG GTT TGA ACT CAG ATC A 56°C Palumbi 1996 16Sar-5' CGC CTG TTT ATC AAA AAC AT - Palumbi 1996 Branch 12S-F1 AAG GAT TTG GCG GTY CTT AAA 52°C This study Branch 12S-R1 GAG CTT AAT TCA AAT TCT YAN TAT TTT - This study Branch 28SF1 GGG TTA AAC GGA TGG ACC TT 56°C This study Branch 28SR1 CGC CTT CGG TCT TTA TCA AC - This study page 4 of 17Zoological Studies 59:14 (2020) © 2020 Academia Sinica, Taiwan 18. AII apex form: [0] entire; [1] bilobed, anterior lobe small; [2] bilobed, posterior lobe small; [3] trilobed. 19. AII apex direction: [0] distally; [1] medially; [2] laterally; [3] posteriolaterally. 20. AII rasp: [0] absent; [1] present. 21. Body sensory papillae: [0] absent; [1] present. 22. Praepipodites: [0] two; [1] one. 23. Endopodites: [0] filtering; [1] female filtering, male scraping; [2] “basket” type; [3] raptorial. 24. Female thoracic dorsolateral projection surface: [0] smooth; [1] papillose; [2] denticulate. 25. Female TH I dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 26. Female TH II dorsal surface: [0] smooth; [1] Medial convexity; [2] Lateral convexity [3] Medial and lateral convexities. 27. Female TH III dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 28. Female TH IV dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 29. Female TH V dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 30. Female TH VI dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 31. Female TH VII dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 32. Female TH VIII dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 33. Female TH IX dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 34. Female TH X dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 35. Female TH XI dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 36. Female TH XII dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 37. Female TH XIII dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 38. Female TH XIV [genital segment] dorsal surface: [0] smooth; [1] medial convexity; [2] lateral convexity [3] medial and lateral convexities. 39. Female abdominal (AB) IV dorsolateral projections: [0] absent; [1] present. 40. Brood pouch: [0] fusiform; [1] pyriform; [2] penduculate; [3] cylindrical; [4] spherical 41. Brood pouch lateral projections: [0] absent; [1] present. 42. Brood pouch extends to: [0] AB I; [1] AB III; [2] AB IV; [3] AB V; [4] AB VI; [5] AB VII 43. Ovaries in thorax: [0] TH 12/13; [1] TH 11; [2] TH 10; [3] TH 9; [4] TH 8; [5] TH 7; [6] TH 6 44. Ovaries in abdomen: [0] none; [1] AB 1; [2] AB 2; [3] AB 3; [4] AB 4; [5] AB 5; [6] AB 6 45. Gonopod ventral projections: [0] none; [1] present. 46. Gonopod distal spined projections: [0] none; [1] one; [2] two. 47. Gonopods extend to: [0] AB I; [1] AB II; [2] AB III. 48. Testicular rami in: [0] abdomen; [1] abdomen and thorax. 49. Testis in abdomen extending to: [0] AB 2; [1] AB 3; [2] AB 4; [3] AB 6. 50. Egg surface: [0] rough; [1] small polygons; [2] medium polygons; [3] large polygons; [4] denticulate. 51. Polygon ridges on egg surface: [0] absent; [1] normal; [2] flanged; [3] spined; [4] obscure. Test for peripatric speciation We tested for peripatric speciation using the approach of Barraclough and Vogler (2000). Briefly species ranges were estimated using museum collection records and published accounts. We concentrated on North American species as our dataset is biased towards these taxa and the occurrence records are better for these species. They were imported into Google Maps to estimate areas. A pruned ultrametric tree was generated from the DNA only dataset using the penalized likelihood algorithm (Sanderson 2002; Kim and Sanderson 2008) implemented in the R package app (Paradise et al. 2004) and clade sequence divergence was estimated using tip to node distances from the ultrametric tree for each clade of interest. Range symmetry, estimated as the range size of the clade with the smaller size divided by the sum of the range sizes of each clade (Barraclough and Vogler 2000), was transformed (arcsin of double the value) and plotted against sequence divergence for each clade. Higher range symmetry values indicate more similar geographic size distributions (e.g., the total area of the ranges is similar between taxa), while lower symmetry values indicate greater differences in geographic size distributions (e.g., the total area of the ranges is dissimilar between taxa—small versus large ranges). Spearman’s rank correlation was used to assess the significance of each relationship. page 5 of 17Zoological Studies 59:14 (2020) © 2020 Academia Sinica, Taiwan RESULTS Phylogenetic analysis DNA sequences for the three gene fragments were obtained for 28 Branchinecta species and the two outgroup species. The sequence evolution model estimation was based on the Bayesian Information Criterion (BIC) in jMODELTEST (Posada 2008). Analysis of each partition indicated the following models were appropriate for each gene fragment: 16S – HKY + I (p = 0.391); 12S – GTR + G (α = 0.296); 28S – GTR + G (α = 0.266). The results of the molecular and molecular/ morphological data analysis gave highly congruent results that supported numerous groups within Branchinecta (Figs. 1, 2). Only posterior probabilities > 0.90 are reported as evidence for strong support. There was limited support for monophyletic groups of species: ‘coloradensis’ group, ‘cornigera’ subgroup, ‘gigas’ group, ‘southern’ group, ‘lindahli’ group, ‘paludosa’ group and ‘sandiegonensis’ subgroup (Fig. 1). The ‘lindahli’ group had low support in the molecules only analysis (0.82) and the ‘packardi’ group had low support in the combined analysis (0.87). The current phylogenetic analyses could not resolve many of the interior branches, though the addition of the morphological data did lead to increased interior branch resolution with low statistical support (Figs. 1, 2). The analyses that included molecules indicated that a trio of species, B. ferox, B. orientalis and B. raptor, were always basal to all other branchinectids analysed here; the two Eurasian species basal to all other branchinectids had low statistical support (Figs. 1, 2). We found 15 equally parsimonious trees with just analyzing morphological data. A 50% majority consensus of these 15 most parsimonious trees had many discrepancies with the trees that included molecular data (Fig. 3). However, there was little to no bootstrap support for any groupings we observed in the molecular datasets. Fig. 1. Bayesian inference phylogeny based on 16S, 12S, 28S and 50 morphological characters. Nodes with filled circles indicate sister group taxon relationships composed of basal broad and narrow range sister taxa. Posterior probabilities: *p = 0.90–0.94; **p = 0.95–0.99; ***p = 1.0. page 6 of 17Zoological Studies 59:14 (2020) © 2020 Academia Sinica, Taiwan We found a significant positive relationship between pairwise clade distance and transformed range symmetry (Fig. 4). This relationship indicates the species which are more closely related have less symmetric (more asymmetric) ranges. SYSTEMATICS Branchinectidae Daday, 1910 (sensu Rogers & Coronel, 2011) Branchipodidae pro partim: Packard 1883 Branchinectidae Daday, 1910; Linder 1941; Tasch 1969; Belk 1982; Brtek & Mura 2000; Belk & Schram 2001; Maeda-Martinez et al. 2002; Rogers & Coronel 2011; Rogers 2013) Diagnosis: Gonopods free and separated, extending ventrolaterally, with rigid base bearing a medial projection. Vas deferens not dorsally looped, lacking clearly defined seminal vesicles. Head lacking a frontal appendage. Second antennae never medially fused, widely separated by labrum, antennal appendages lacking. Male proximal antennomere often spinose, dentate and/ or tuberculate medially, and/or posteriorly, pulvilli and apophyses present or not. Second maxilla robust, elongated, well developed, with 5–30 apical rigid setae, and 3–5 anteriorly directed soft setae on the anteriomedial surface. Eleven pairs of thoracopods with each thoracopod bearing one prae-epipodite. Female thoracic segments may have rows or paired rows of lateral and/or dorsal projections. Body length ranging from 6 to 100+ mm. Two genera. Archaebranchinecta Rogers & Coronel, in Rogers 2019 Branchinecta Daday, 1910: Harding 1940; Belk & Brtek 1995; Brtek & Mura 2000; Belk & Schram 2001 Archaebranchinecta Rogers & Coronel, 2011; Rogers 2013; Rogers 2019; Cohen et al. 2019 Diagnosis: Genital segments expanded, gonopod rigid bases extending to base of abdominal segment II, Fig. 2. Bayesian inference phylogeny based on 16S, 12S, and 28S. Nodes with filled circles indicate sister taxon relationships composed of basal broad and narrow range sister taxa. Posterior probabilities: *p = 0.90–0.94; **p = 0.95–0.99; ***p = 1.0. page 7 of 17Zoological Studies 59:14 (2020) © 2020 Academia Sinica, Taiwan and bearing a single proximomedial fleshy lobe and chitinized medial projection, terminating in biramal structure. Biramal structure proximal ramus conical and subacute. Distal ramus arcuate, spiniform, directed medially, connected to proximal ramus by a ridge. Gonopod eversible portion with one anterior and one lateral longitudinal denticle row. Apical most portion hemispherical, situated in recess with ten to twelve anterioapically scattered acute spinules. Abdominal segment I bearing a ventral chitinous plate covered in denticles. Three species are included in this genus, and Archaebranchinecta pollicifera (Harding 1940) is the type by designation (Rogers 2019). Species attributed Archaebranchinecta aimara Cohen et al. 2019 †Archaebranchinecta barstowensis (Belk & Schram, 2001) = †Branchinecta barstowensis Belk & Schram, 2001 Archaebranchinecta pollicifera (Harding, 1940) (sensu Rogers & Coronel, 2011) = Branchinecta pollicifera Harding, 1940 Branchinecta Müller, 1788 (sensu Rogers & Coronel, 2011) Branchinecta Müller, 1788; Packard 1874, 1883; Verrill 1869; Shantz 1905; Daday 1910; Linder 1941; Belk & Brtek 1995; Brtek & Mura 2000; Belk & Schram 2001; Maeda-Martinez et al. 2002; Rogers & Coronel 2011; Rogers 2013 Artemis Thompson, 1834 Branchiopsyllus Sars, 1897; Linder 1941; Belk 1982 (fide Vekhov 1989) Artemiella Daday, (fide Linder 1932, 1941) Diagnosis: Genital segments not expanded, each gonopod extending ventrolaterally, visible in dorsal view, with rigid base bearing a spiniform medial projection. Gonopod eversible portion with one or two denticulate tubercles apically or subapically. Apex truncated. Abdominal segment I lacking a ventral Fig. 3. 50% Majority consensus tree from the 15 most parsimonious trees produced from a heuristic search of 51 morphological characters in PAUP. The proportion of 1,000 bootstrap replicates above 70% are shown. page 8 of 17Zoological Studies 59:14 (2020) © 2020 Academia Sinica, Taiwan chitinized plate. Brood pouch variable, may be elongate, pedunculate, conic or pyriform. Females may have corneous or papillose cephalic projections. Type by monotypy Branchinecta paludosa (Müller, 1788). Comments: There are 52 species presently recognized (Rogers 2013; Rogers and Lorenz 2015) in seven species groups. Due to the lack diagnosis or definition in the work of Brtek and Mura (2000), we have made no effort to associate our species groups with theirs; any attempt would be pure speculation. Subdivision: All the following species groups and incertae sedis taxa are part of Branchinecta. The shared characters between the ferox and the raptor groups, and the fact that these groups were consistently basal in our analyses, suggest that Branchinecta may have had a Eurasian origin, and via a stochastic dispersal event, invaded North America and rapidly differentiated across that continent in a manner similar to Streptocephalus (Daniels et al. 2004). “ferox” species group Diagnosis: Eyes normal; male second antennae arcuate and subacute; female second antennae lacking a medial spine; body lacking sensory papillae; female thoracic segments lacking any dorsolateral projections; first and last limb pairs shortest; ovaries uniramal limited to the abdomen (except B. ferox, which is biramal, extending anteriorly to thoracic segment X), and posteriorly in the abdomen to abdominal segment IV, V, or VI; brood pouch fusiform, extending to abdominal segment III, IV, V or VI; testes always uniramal extending posteriorly from genital segments into abdominal segment IV, V, or VI; cercopods cylindrical, tapering, shorter than the last two abdominal segments, medial surface with setae, lateral surface with or without setae. No geochemical association data is available. Comments: Three species, morphologically similar to B. raptor in the overall general form of the male second antennae and the cylindrical cercopods. However, in B. raptor the cercopods are glabrous, whereas in the ferox group, marginal setae are always present medially in all three species, but on the lateral side may only be present distally or not at all in B. ferox and B. orientalis. Species attributed Branchinecta ferox (Milne-Edwards, 1840) (sensu Petkovski, 1991) = Branchipus ferox Milne-Edwards, 1840 = Branchipus eximius Baird, 1861 = Branchipus (Branchinecta) ferus Brauer, 1877 = Branchipus ferox f. aestivalis Daday, 1890 = Branchipus ferox f. hibernalis Daday, 1890 = Branchipus ferox f. vernalis Daday, 1890 Branchinecta minuta Smirnov, 1948 Branchinecta orientalis Sars, 1901 (sensu Petkovski, 1991) = Branchinecta cervantesi Margalef, 1947 = Branchinecta ferox orientalis Sars, 1901 “raptor” species group Diagnosis: Eyes reduced; male second antennae elongate, arcuate and subacute; female second antennae lacking a medial spine; body covered in sensory papillae; female thoracic segments lacking any dorsolateral projections; anterior most limbs longest; ovaries uniramal limited to the abdomen; brood pouch pyriform, extending to abdominal segment III; testes uniramal extending posteriorly from genital segments into abdominal segment VI; cercopods elongate, nearly as long as the abdomen, cylindrical, tapering, and glabrous. All other anostracans have the first and last limb pairs smaller than the middle pairs. This species is only known from habitats with a substrate salinity of 5 mS/cm and containing 25% calcium carbonate (Rogers 2014a). Comments: Branchinecta raptor shares several important character states with the ferox group (see comments under the ferox group below) and with Branchinecta gigas in the gigas group. Both B. raptor and B. gigas are specialized predators. Both species are super giants among anostracans (growing over 100 mm), have dramatically reduced eyes, bodies covered in sensory papillae that are used to detect movement in water by potential prey items, and elongated, whip like cercopods (Rogers et al. 2006). The significance of these characters must needs be examined, especially Fig. 4. Relationship between pairwise clade distance and transformed range symmetry. 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