1 Title: Revealing paraphyly and placement of extinct species within Epischura (Copepoda: 2 Calanoida) using molecular data and quantitative morphometrics 3 4 Author List: Larry L. Bowman, Jr.1,2, Daniel J. MacGuigan1, Madeline E. Gorchels3,4, Madeline 5 M. Cahillane3,5, and Marianne V. Moore3 6 7 1 Department of Ecology and Evolutionary Biology, Yale University, Osborn Memorial 8 Laboratories, 165 Prospect St., New Haven, CT, USA 06511 9 2Corresponding author: [email protected] 10 3Department of Biological Sciences, Wellesley College, 106 Central St., Wellesley, MA, USA 11 02481-0832 12 4Current address: Bren School of Environmental Science & Management, Bren Hall, 2400, 13 University of California, Santa Barbara, CA, USA 93117 14 5384 South St., Northampton, MA 01060 15 16 Declarations of interest: none. Genetics Epischura (Epischurella) baikalensis Epischura (Epischurella) chankensis Heterocope septentrionalis Diaptomidae Epischura fluviatilis .94 Epischura nordenskioldi Pseudodiaptomidae Epischura lacustris Temoridae* .99 Epischura nevadensis Fosshageniidae .96 Bathypontiidae .83 Temora discaudata .58 .97 .98 Sulcanidae Temora longicornis .97 .83 125 100 75 50 25 0 MYBP Rhincalanidae Centropagidae .82 Morphology Epischura lacustris Eucalanidae .88 .88 Pontellidae .89 Tortanidae Temora discaudata .72 Candaciidae Epischura nevadensis Subeucalanidae .64 .73 .39 Epischura nordenskioldi .66 Temoridae* .79 Epischura fluviatilis Temora longicornis .83 250 150 50 MYBP .93 Epischura (Epischurella) baikalensis .97 Epischura (Epischurella) chankensis Spinocalanidae Epischura massachusettsensis .98 .94 .90 Heterocope septentrionalis .87 .67 .93 .44 .89 Calanidae .77 Genetics + Morphology Epischura (Epischurella) baikalensis Tharybidae .86 .52 Epischura (Epischurella) chankensis Phaennidae .64 Diaixidae .58 Epischura massachusettsensis .57 Heterocope septentrionalis .97 .79 Scolecitrichidae .98 Epischura fluviatilis .89 Epischura nordenskioldi .98 Clausocalanidae Epischura lacustris Paracalanidae Aetideidae* Epischura nevadensis Euchaetidae* Aetideidae* Temora discaudata .84 Euchaetidae* Temora longicornis Megacalanidae 125 100 75 50 25 0 MYBP 17 Abstract 18 Epischura (Calanoida: Temoridae) is a Holarctic group of copepods serving important ecological 19 roles, but it is difficult to study because of small range sizes of individual species and widespread 20 distribution of the genus. This genus includes Tertiary relicts, some endemic to single, isolated 21 lakes and can play major roles in unique ecosystems like Lakes Baikal and Tahoe. We present 22 the first molecular and morphological analysis of Epischura that reveals their spatio-temporal 23 evolutionary history. Morphological measurements of mandibles and genetics estimated 24 phylogenetic relationships among all species represented in Epischura, including E. 25 massachusettsensis, whose extinction status is of concern. Analyses used three gene regions for 26 six previously unsequenced species to infer highly-resolved and well-supported phylogenies 27 confirming a split between Siberian and North American species. Previously published age 28 estimates and sequence data from broad taxonomic sampling of calanoid copepods estimated 29 divergence times between the two Epischura groups. Divergence time estimates for Epischura 30 were consistent with earlier molecular clock estimates and late-Miocene cooling events. 31 Additionally, we provide the first taxonomically broad estimates of divergence times within 32 Calanoida. The paraphyletic nature of the genus Epischura (and the family Temoridae) is 33 apparent and requires the resurrection of the genus Epischurella (Smirnov 1936) to describe the 34 Siberian species. 35 36 1. Introduction 37 38 As a group, Epischura (Calanoida:Temoridae) copepods inhabit the entire Holarctic 39 (Figure 1). Given their disjunct distribution across the Holarctic and small range sizes, 40 Epischura may represent one of the few animal examples of Tertiary relict endemism, also noted 41 in many plant species (Wu, et al, 2005). Yet, having survived in endemic refugia for millions of 42 years, the Epischura may be at risk given the rapid pace of climate change in the Holarctic zone. 43 Notably, Epischura baikalensis is currently listed as an IUCN threatened species due to pollution 44 and E. massachusettsensis (possibly extinct) has not been observed since the 1950s (Humes, 45 1955)—making them both of conservation concern. While most species in the group, namely, E. 46 baikalensis, E. chankensis, E. nevadensis, E. massachusettsensis, and to a lesser extent, E. 47 nordenskioldi, are geographically isolated to single or few proximal lakes, there are also two 48 widespread species, E. lacustris and E. fluviatilis that regularly establish in reservoirs (Bowman, 49 T. E., 1991; DeBiase and Taylor, 1993). Two of the species, E. baikalensis and E. chankensis, 50 are endemic to Siberia, and the five remaining species are endemic to North America, so 51 understanding the timing and relationship of this divergence will give insight into the history of 52 the group’s Holarctic distribution. 53 Dispersal in obligate, sexually-reproducing zooplankton is likely to be extremely limited 54 (Allen, 2007), restricting the ability of Epischura to migrate or establish in new habitats. 55 Consequently, it is imperative to understand the evolutionary history and genetic diversity within 56 and among this vulnerable group. Epischura are also ecologically important as aggressive 57 primary and secondary consumers, often serving as critical links between trophic levels in the 58 foodwebs in which they inhabit (Moore, et al, 2019). In general, Epischura reside in the pelagic 59 zone of lakes that lack a diverse zooplanktivorous fish assemblage but have a robust community 60 of small-bodied zooplankton (Brooks and Dodson, 1965). Hence, we see Epischura in Lakes 61 Baikal and Tahoe and large reservoirs in the Southeastern USA. However, we also find them in 62 smaller-volume ecosystems, such as vernal pools and kettle ponds in New England and Lake 63 Khanka on the Eastern Siberian-Chinese border (Ma, et al, 2019), where they likely serve as 64 keystone consumers. Due to their expansive geographic distribution and restricted endemism 65 (Afanasyeva, 1998), few studies have explored the group as a whole. 66 Here we performed the first phylogenetic and morphological analyses of Epischura in an 67 effort to understand their evolutionary history and to evaluate their relationships to each other 68 and within the Temoridae. Using another Holarctic sister genus Heterocope as an outgroup, we 69 constructed a dataset that includes three rRNA gene regions across all extant species represented 70 in the genus. In addition, we used publicly available data to reconstruct the evolutionary history 71 of Calanoida, the order containing Epischura and around 1,800 other species. We show that 72 Epischura is a paraphyletic group, interspersed with the genus Heterocope. We suggest that the 73 Siberian species constitute their own genus, diverging from the North American species 74 ~40MYBP, and we propose resurrecting the originally assigned genus name, Epischurella 75 (Smirnov, 1936). 76 77 78 2. Materials and methods 79 80 2.1 Sample collection, DNA extraction, and sequencing 81 82 We generated DNA sequences for seven species of calanoid copepods, including six from 83 the genus Epischura and one outgroup from the genus Heterocope (Table 1and Supplemental 84 Table 1). All specimens are vouched at the Smithsonian National Museum of Natural History 85 Invertebrate Zoology Collections: E. baikalensis (USNM 1578660), E. chankensis (USNM 86 1578661), E. fluviatilis (USNM 1578664), E. lacustris (1578662), E. nevadensis (USNM 87 1578665), E. nordenskioldi (USNM 1578663), H. septentrionalis (USNM 1578666). All 88 specimens were collected from the field (Figure 1) directly via plankton net sampling between 89 2012 and 2016 and preserved in 70% ethanol until extraction, with the exception of E. 90 massachusettsensis. Formalin-fixed E. massachusettsensis specimens were acquired from the 91 Smithsonian National Museum of Natural History Invertebrate Zoology Collections (USNM 92 93932). We were unable to extract or amplify DNA from E. massachusettsensis specimens 93 despite multiple attempts with various protocols including a standard extraction protocol 94 (Bowman Jr, et al, 2017), a Qiagen DNeasy blood and tissue kit (Qiagen, Inc. Valencia, CA), a 95 modified ancient DNA protocol (Ruane and Austin, 2017), and phenol-chloroform extraction 96 (Pine, et al, 1999). 97 Animals were identified under a 40x Leica dissecting microscope and vouchered before 98 extraction. We used the DNA extraction protocol outlined in Bowman et al. (2017) to extract 99 DNA, which had been previously shown to work well with the Epischura group (Bowman Jr, et 100 al, 2017). This extraction protocol involves homogenizing whole-body specimens in 25uL of 101 50mM TE buffer, pH 8.0 that contains 60mAU/mL proteinase K (Qiagen) before incubation at 102 54èC for 20min. For Epischura nordenskioldi and Epischura nevadensis, we used the Qiagen 103 DNeasy blood and tissue kit per the manufacturer’s directions, due to low-yield results with the 104 aforementioned protocol. 105 Three regions of ribosomal DNA were sequenced: 18s, ITS2, and 28s. We used oligo 106 primers found in the literature for amplification with their published PCR protocols (Table 2) 107 except for Epischura nordenskioldi, for which we designed a species-specific primer